1,2,5-Oxadiazoles As Inhibitors Of Indoleamine 2,3- Dioxygenase

ABSTRACT

The present invention is directed to 1,2,5-oxadiazole derivatives, and compositions of the same, which are inhibitors of indoleamine 2,3-dioxygenase and are useful in the treatment of cancer and other disorders, and to the processes and intermediates for making such 1,2,5-oxadiazole derivatives.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/322,362, filedJul. 2, 2014, which is a continuation of U.S. Ser. No. 13/294,711, filedNov. 11, 2011, which is a divisional of U.S. Ser. No. 12/498,782, filedJul. 7, 2009, which claims the benefit of U.S. Ser. No. 61/078,876,filed Jul. 8, 2008 and U.S. Ser. No. 61/150,873, filed Feb. 9, 2009, thedisclosures of each of which are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention is directed to 1,2,5-oxadiazole derivatives whichare inhibitors of indoleamine 2,3-dioxygenase and are useful in thetreatment of cancer and other disorders, and to processes andintermediates for making the same.

BACKGROUND OF THE INVENTION

Tryptophan (Trp) is an essential amino acid required for thebiosynthesis of proteins, niacin and the neurotransmitter5-hydroxytryptamine (serotonin). The enzyme indoleamine 2,3-dioxygenase(also known as INDO or IDO) catalyzes the first and rate limiting stepin the degradation of L-tryptophan to N-formyl-kynurenine. In humancells, a depletion of Trp resulting from IDO activity is a prominentgamma interferon (IFN-γ)-inducible antimicrobial effector mechanism.IFN-γ stimulation induces activation of IDO, which leads to a depletionof Trp, thereby arresting the growth of Trp-dependent intracellularpathogens such as Toxoplasma gondii and Chlamydia trachomatis. IDOactivity also has an antiproliferative effect on many tumor cells, andIDO induction has been observed in vivo during rejection of allogeneictumors, indicating a possible role for this enzyme in the tumorrejection process (Daubener, et al., 1999, Adv. Exp. Med. Biol., 467:517-24; Taylor, et al., 1991, FASEB J., 5: 2516-22).

It has been observed that HeLa cells co-cultured with peripheral bloodlymphocytes (PBLs) acquire an immuno-inhibitory phenotype throughup-regulation of IDO activity. A reduction in PBL proliferation upontreatment with interleukin-2 (IL2) was believed to result from IDOreleased by the tumor cells in response to IFNG secretion by the PBLs.This effect was reversed by treatment with 1-methyl-tryptophan (1MT), aspecific IDO inhibitor. It was proposed that IDO activity in tumor cellsmay serve to impair antitumor responses (Logan, et al., 2002,Immunology, 105: 478-87).

Recently, an immunoregulatory role of Trp depletion has received muchattention. Several lines of evidence suggest that IDO is involved ininduction of immune tolerance.

Studies of mammalian pregnancy, tumor resistance, chronic infections andautoimmune diseases have shown that cells expressing IDO can suppressT-cell responses and promote tolerance. Accelerated Trp catabolism hasbeen observed in diseases and disorders associated with cellular immuneactivation, such as infection, malignancy, autoimmune diseases and AIDS,as well as during pregnancy. For example, increased levels of IFNs andelevated levels of urinary Trp metabolites have been observed inautoimmune diseases; it has been postulated that systemic or localdepletion of Trp occurring in autoimmune diseases may relate to thedegeneration and wasting symptoms of these diseases. In support of thishypothesis, high levels of IDO were observed in cells isolated from thesynovia of arthritic joints. IFNs are also elevated in humanimmunodeficiency virus (HIV) patients and increasing IFN levels areassociated with a worsening prognosis. Thus, it was proposed that IDO isinduced chronically by HIV infection, and is further increased byopportunistic infections, and that the chronic loss of Trp initiatesmechanisms responsible for cachexia, dementia and diarrhea and possiblyimmunosuppression of AIDS patients (Brown, et al., 1991, Adv. Exp. Med.Biol., 294: 425-35). To this end, it has recently been shown that IDOinhibition can enhance the levels of virus-specific T cells and,concomitantly, reduce the number of virally-infected macrophages in amouse model of HIV (Portula et al., 2005, Blood, 106: 2382-90).

IDO is believed to play a role in the immunosuppressive processes thatprevent fetal rejection in utero. More than 40 years ago, it wasobserved that, during pregnancy, the genetically disparate mammalianconceptus survives in spite of what would be predicted by tissuetransplantation immunology (Medawar, 1953, Symp. Soc. Exp. Biol. 7:320-38). Anatomic separation of mother and fetus and antigenicimmaturity of the fetus cannot fully explain fetal allograft survival.Recent attention has focused on immunologic tolerance of the mother.Because IDO is expressed by human syncytiotrophoblast cells and systemictryptophan concentration falls during normal pregnancy, it washypothesized that IDO expression at the maternal-fetal interface isnecessary to prevent immunologic rejection of the fetal allografts. Totest this hypothesis, pregnant mice (carrying syngeneic or allogeneicfetuses) were exposed to 1MT, and a rapid, T cell-induced rejection ofall allogeneic concepti was observed. Thus, by catabolizing tryptophan,the mammalian conceptus appears to suppresses T-cell activity anddefends itself against rejection, and blocking tryptophan catabolismduring murine pregnancy allows maternal T cells to provoke fetalallograft rejection (Munn, et al., 1998, Science, 281: 1191-3).

Further evidence for a tumoral immune resistance mechanism based ontryptophan degradation by IDO comes from the observation that most humantumors constitutively express IDO, and that expression of IDO byimmunogenic mouse tumor cells prevents their rejection by preimmunizedmice. This effect is accompanied by a lack of accumulation of specific Tcells at the tumor site and can be partly reverted by systemic treatmentof mice with an inhibitor of IDO, in the absence of noticeable toxicity.Thus, it was suggested that the efficacy of therapeutic vaccination ofcancer patients might be improved by concomitant administration of anIDO inhibitor (Uyttenhove et al., 2003, Nature Med., 9: 1269-74). It hasalso been shown that the IDO inhibitor, 1-MT, can synergize withchemotherapeutic agents to reduce tumor growth in mice, suggesting thatIDO inhibition may also enhance the anti-tumor activity of conventionalcytotoxic therapies (Muller et al., 2005, Nature Med., 11: 312-9).

One mechanism contributing to immunologic unresponsiveness toward tumorsmay be presentation of tumor antigens by tolerogenic host APCs. A subsetof human IDO-expressing antigen-presenting cells (APCs) that coexpressedCD123 (IL3RA) and CCR6 and inhibited T-cell proliferation have also beendescribed. Both mature and immature CD123-positive dendritic cellssuppressed T-cell activity, and this IDO suppressive activity wasblocked by 1MT (Munn, et al., 2002, Science, 297: 1867-70). It has alsobeen demonstrated that mouse tumor-draining lymph nodes (TDLNs) containa subset of plasmacytoid dendritic cells (pDCs) that constitutivelyexpress immunosuppressive levels of IDO. Despite comprising only 0.5% oflymph node cells, in vitro, these pDCs potently suppressed T cellresponses to antigens presented by the pDCs themselves and also, in adominant fashion, suppressed T cell responses to third-party antigenspresented by nonsuppressive APCs. Within the population of pDCs, themajority of the functional IDO-mediated suppressor activity segregatedwith a novel subset of pDCs coexpressing the B-lineage marker CD19.Thus, it was hypothesized that IDO-mediated suppression by pDCs in TDLNscreates a local microenvironment that is potently suppressive of hostantitumor T cell responses (Munn, et al., 2004, J. Clin. Invest.,114(2): 280-90).

IDO degrades the indole moiety of tryptophan, serotonin and melatonin,and initiates the production of neuroactive and immunoregulatorymetabolites, collectively known as kynurenines. By locally depletingtryptophan and increasing proapoptotic kynurenines, IDO expressed bydendritic cells (DCs) can greatly affect T-cell proliferation andsurvival. IDO induction in DCs could be a common mechanism of deletionaltolerance driven by regulatory T cells. Because such tolerogenicresponses can be expected to operate in a variety of physiopathologicalconditions, tryptophan metabolism and kynurenine production mightrepresent a crucial interface between the immune and nervous systems(Grohmann, et al., 2003, Trends Immunol., 24: 242-8). In states ofpersistent immune activation, availability of free serum Trp isdiminished and, as a consequence of reduced serotonin production,serotonergic functions may also be affected (Wirleitner, et al., 2003,Curr. Med. Chem., 10: 1581-91).

Interestingly, administration of interferon-α has been observed toinduce neuropsychiatric side effects, such as depressive symptoms andchanges in cognitive function. Direct influence on serotonergicneurotransmission may contribute to these side effects. In addition,because IDO activation leads to reduced levels of tryptophan, theprecursor of serotonin (5-HT), IDO may play a role in theseneuropsychiatric side effects by reducing central 5-HT synthesis.Furthermore, kynurenine metabolites such as 3-hydroxy-kynurenine(3-OH-KYN) and quinolinic acid (QUIN) have toxic effects on brainfunction. 3-OH-KYN is able to produce oxidative stress by increasing theproduction of reactive oxygen species (ROS), and QUIN may produceoverstimulation of hippocampal N-methyl-D-aspartate (NMDA) receptors,which leads to apoptosis and hippocampal atrophy. Both ROSoverproduction and hippocampal atrophy caused by NMDA overstimulationhave been associated with depression (Wichers and Maes, 2004, J.Psychiatry Neurosci., 29: 11-17). Thus, IDO activity may play a role indepression.

Small molecule inhibitors of IDO are being developed to treat or preventIDO-related diseases such as those described above. For example,oxadiazole and other heterocyclic IDO inhibitors are reported in US2006/0258719 and US 2007/0185165. PCT Publication WO 99/29310 reportsmethods for altering T cell-mediated immunity comprising altering localextracellular concentrations of tryptophan and tryptophan metabolites,using an inhibitor of IDO such as 1-methyl-DL-tryptophan,p-(3-benzofuranyl)-DL-alanine, p-[3-benzo(b)thienyl]-DL-alanine, and6-nitro-L-tryptophan) (Munn, 1999). Reported in WO 03/087347, alsopublished as European Patent 1501918, are methods of makingantigen-presenting cells for enhancing or reducing T cell tolerance(Munn, 2003). Compounds having indoleamine-2,3-dioxygenase (IDO)inhibitory activity are further reported in WO 2004/094409; and U.S.Patent Application Publication No. 2004/0234623 is directed to methodsof treating a subject with a cancer or an infection by theadministration of an inhibitor of indoleamine-2,3-dioxygenase incombination with other therapeutic modalities.

In light of the experimental data indicating a role for IDO inimmunosuppression, tumor resistance and/or rejection, chronicinfections, HIV-infection, AIDS (including its manifestations such ascachexia, dementia and diarrhea), autoimmune diseases or disorders (suchas rheumatoid arthritis), and immunologic tolerance and prevention offetal rejection in utero, therapeutic agents aimed at suppression oftryptophan degradation by inhibiting IDO activity are desirable.Inhibitors of IDO can be used to activate T cells and therefore enhanceT cell activation when the T cells are suppressed by pregnancy,malignancy or a virus such as HIV. Inhibition of IDO may also be animportant treatment strategy for patients with neurological orneuropsychiatric diseases or disorders such as depression. Thecompounds, compositions and methods herein help meet the current needfor IDO modulators.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, IDO inhibitors of Formula I:

or pharmaceutically acceptable salts thereof, wherein constituentvariables are defined herein.

The present invention further provides a pharmaceutical compositioncomprising a compound of Formula I, and at least one pharmaceuticallyacceptable carrier.

The present invention further provides a method of inhibiting activityof indoleamine 2,3-dioxygenase comprising contacting the indoleamine2,3-dioxygenase (IDO) with a compound of Formula I, or apharmaceutically acceptable salt thereof.

The present invention further provides a method of inhibitingimmunosuppression in a patient comprising administering to said patientan effective amount of a compound of Formula I, or a pharmaceuticallyacceptable salt thereof.

The present invention further provides a method of treating cancer,viral infection, depression, a neurodegenerative disorder, trauma,age-related cataracts, organ transplant rejection, or an autoimmunedisease in a patient comprising administering to said patient atherapeutically effective amount of a compound of Formula I, or apharmaceutically acceptable salt thereof.

The present invention further provides a method of treating melanoma ina patient comprising administering to said patient a therapeuticallyeffective amount of a compound of Formula I, or a pharmaceuticallyacceptable salt thereof.

The present invention further provides intermediates, processes ofpreparing the same, and compositions containing the same, which areuseful in the preparation of a compound of Formula F15:

The present invention further provides intermediates, processes ofpreparing the same, and compositions containing the same, which areuseful in the preparation of a compound of Formula F19:

The present invention further provides intermediates, processes ofpreparing the same, and compositions containing the same, which areuseful in the preparation of a compound of Formula F28:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an XRPD pattern characteristic of the compound of theinvention prepared in Example 1.

FIG. 2 shows a DSC thermogram characteristic of the compound of theinvention prepared in Example 1.

FIG. 3 shows TGA data characteristic of the compound of the inventionprepared in Example 1.

DETAILED DESCRIPTION

The present invention provides, inter alia, IDO inhibitors of Formula I:

or pharmaceutically acceptable salts thereof, wherein:

R¹ is NH₂ or CH₃;

R² is Cl, Br, CF₃, CH₃, or CN;

R³ is H or F; and

n is 1 or 2.

In some embodiments, R¹ is NH₂.

In some embodiments, R¹ is CH₃.

In some embodiments, R² is Cl.

In some embodiments, R² is Br.

In some embodiments, R² is CF₃.

In some embodiments, R² is CH₃.

In some embodiments, R² is CN.

In some embodiments, R³ is H.

In some embodiments, R³ is F.

In some embodiments, n is 1.

In some embodiments, n is 2.

The compounds of the present invention can exist in various solid forms.As used herein “solid form” is meant to refer to a solid characterizedby one or more properties such as, for example, melting point,solubility, stability, crystallinity, hygroscopicity, water content, TGAfeatures, DSC features, DVS features, XRPD features, etc. Solid forms,for example, can be amorphous, crystalline, or mixtures thereof.

Different crystalline solid forms typically have different crystallinelattices (e.g., unit cells) and, usually as a result, have differentphysical properties. In some instances, different crystalline solidforms have different water or solvent content. The different crystallinelattices can be identified by solid state characterization methods suchas by X-ray powder diffraction (XRPD). Other characterization methodssuch as differential scanning calorimetry (DSC), thermogravimetricanalysis (TGA), dynamic vapor sorption (DVS), and the like further helpidentify the solid form as well as help determine stability andsolvent/water content.

In one aspect, the present invention provides various solid forms of4-({2-[(aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide(see Example 1). In some embodiments, the solid form is a crystallinesolid. In some embodiments, the solid form is substantially anhydrous(e.g., contains less than about 1% water, less than about 0.5% water,less than about 1.5% water, less than about 2% water,). In someembodiments, the solid form is characterized by a melting point of, or aDSC endotherm centered at, about 162 to about 166° C. In someembodiments, the solid form is characterized by a melting point of, or aDSC endotherm centered at, about 164° C. In some embodiments, the solidform has a DSC thermogram substantially as shown in FIG. 2. In furtherembodiments, the solid form has at least one, two or three XRPD peaks,in terms of 2-theta, selected from about 18.4°, about 18.9°, about21.8°, about 23.9°, about 29.2°, and about 38.7°. In furtherembodiments, the solid form has an XRPD pattern substantially as shownin FIG. 1.

The present invention further provides a composition comprising a solidform of4-({2-[(aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide(see Example 1). The composition can comprise at least about 50%, atleast about 75%, at least about 90%, at least about 95%, or at leastabout 99% by weight of the solid form. The composition can also containa pharmaceutically acceptable excipient. In some embodiments, the solidform is substantially purified.

An XRPD pattern of reflections (peaks) is typically considered afingerprint of a particular crystalline form. It is well known that therelative intensities of the XRPD peaks can widely vary depending on,inter alia, the sample preparation technique, crystal size distribution,various filters used, the sample mounting procedure, and the particularinstrument employed. In some instances, new peaks may be observed orexisting peaks may disappear, depending on the type of the instrument orthe settings. As used herein, the term “peak” refers to a reflectionhaving a relative height/intensity of at least about 4% of the maximumpeak height/intensity. Moreover, instrument variation and other factorscan affect the 2-theta values. Thus, peak assignments, such as thosereported herein, can vary by plus or minus about 0.2° (2-theta), and theterm “substantially” as used in the context of XRPD herein is meant toencompass the above-mentioned variations.

In the same way, temperature readings in connection with DSC, TGA, orother thermal experiments can vary about ±3° C. depending on theinstrument, particular settings, sample preparation, etc. Accordingly, acrystalline form reported herein having a DSC thermogram “substantially”as shown in any of the Figures is understood to accommodate suchvariation.

At various places in the present specification, substituents ofcompounds of the invention may be disclosed in groups or in ranges. Itis specifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges.

It is intended that the compounds of the invention are stable. As usedherein “stable” refers to a compound that is sufficiently robust tosurvive isolation to a useful degree of purity from a reaction mixture,and preferably capable of formulation into an efficacious therapeuticagent.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

The compounds of the invention are further intended to include allpossible geometric isomers. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms. A bond in astructure diagram represented by a wavy line “

” is intended to indicate that the structure represents the cis or thetrans isomer, or a mixture of the cis and trans isomers in anyproportion.

Compounds of the invention also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

In some embodiments, the compounds of the invention, and salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compound of theinvention. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compound of the invention, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The present invention also includes salts of the compounds describedherein. As used herein, “salts” refers to derivatives of the disclosedcompounds wherein the parent compound is modified by converting anexisting acid or base moiety to its salt form. Examples of saltsinclude, but are not limited to, mineral acid (such as HCl, HBr, H₂SO₄)or organic acid (such as acetic acid, benzoic acid, trifluoroaceticacid) salts of basic residues such as amines; alkali (such as Li, Na, K,Mg, Ca) or organic (such as trialkylammonium) salts of acidic residuessuch as carboxylic acids; and the like. The salts of the presentinvention can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile (ACN) are preferred.

The “pharmaceutically acceptable salts” of the present invention includea subset of the “salts” described above which are, conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. Lists of suitable salts are foundin Remington's Pharmaceutical Sciences, 17^(th) ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418 and Journal of PharmaceuticalScience, 66, 2 (1977), each of which is incorporated herein by referencein its entirety. The phrase “pharmaceutically acceptable” is employedherein to refer to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Methods of Synthesis

The compounds of the present invention can be prepared in a variety ofways known to one skilled in the art of organic synthesis. The compoundsof the present invention can be synthesized using the methods ashereinafter described below, together with synthetic methods known inthe art of synthetic organic chemistry or variations thereon asappreciated by those skilled in the art.

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given; other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C), infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry; or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography. The compounds obtained by the reactions can be purifiedby any suitable method known in the art. For example, chromatography(medium pressure) on a suitable adsorbent (e.g., silica gel, alumina andthe like) HPLC, or preparative thin layer chromatography; distillation;sublimation, trituration, or recrystallization.

Preparation of compounds can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Wuts and Greene, Greene's Protective Groups inOrganic Synthesis, 4^(th) Ed., John Wiley & Sons: New York, 2006, whichis incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the reaction step, suitable solvent(s) for that particularreaction step can be selected. Appropriate solvents include water,alkanes (such as pentanes, hexanes, heptanes, cyclohexane, etc., or amixture thereof), aromatic solvents (such as benzene, toluene, xylene,etc.), alcohols (such as methanol, ethanol, isopropanol, etc.), ethers(such as dialkylethers, methyl tert-butyl ether (MTBE), tetrahydrofuran(THF), dioxane, etc.), esters (such as ethyl acetate, butyl acetate,etc.), halogenated solvents (such as dichloromethane (DCM), chloroform,dichloroethane, tetrachloroethane), dimethylformamide (DMF),dimethylsulfoxide (DMSO), acetone, acetonitrile (ACN),hexamethylphosphoramide (HMPA) and N-methyl pyrrolidone (NMP). Suchsolvents can be used in either their wet or anhydrous forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includes fractionalrecrystallization using a “chiral resolving acid” which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids. Resolution ofracemic mixtures can also be carried out by elution on a column packedwith an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

The compounds of the invention can be prepared, for example, using thereaction pathways and techniques as described below.

The processes and intermediates of the present invention are useful inthe preparation of IDO inhibitors. A general scheme for the preparationof compounds F15 of the invention are described in Scheme 1.

Referring now to Scheme 1, the invention provides a process forpreparing a compound of Formula F15, or a salt thereof, wherein R² isCl, Br, CF₃, CH₃, or CN; R³ is H or F; and n is 1 or 2, by reacting acompound of Formula F13, or a salt thereof, wherein Pg¹ is an aminoprotecting group, with an amino deprotecting agent (Step M) to afford acompound of Formula F14, or a salt thereof; and reacting the compound ofFormula F14 with a base (Step N) to afford the compound of Formula F15.The compound of Formula F15 can be purified by trituration orrecrystallization using solvents such as water, ethanol, MTBE or acombination thereof.

In some embodiments, R² is Br, R³ is F, and n is 1.

In some embodiments, R² is Br, R³ is F, and n is 2.

In some embodiments, R² is Cl, R³ is F, and n is 1.

In some embodiments, R² is Cl, R³ is F, and n is 2.

In some embodiments, R² is CF₃, R³ is F, and n is 1.

In some embodiments, R² is CF₃, R³ is F, and n is 2.

In some embodiments, R² is CF₃, R³ is H, and n is 1.

In some embodiments, R² is CF₃, R³ is H, and n is 2.

In some embodiments, R² is CN, R³ is F, and n is 1.

Amino protecting groups are regularly used in organic synthesis toprevent unwanted reactions of an amino group while performing a desiredtransformation. Amino protecting groups allow easy covalent attachmentto a nitrogen atom as well as selective cleavage from the nitrogen atom.Various amino protecting groups, classified broadly as alkoxycarbonyl(such as ethoxycarbonyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl(Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), and the like), acyl (such asacetyl (Ac), benzoyl (Bz), and the like), sulfonyl (such asmethanesulfonyl, trifluoromethanesulfonyl, and the like), arylalkyl(such as benzyl, diphenylmethyl, triphenylmethyl (trityl), and thelike), alkenylalkyl (such as allyl, prenyl, and the like),diarylmethyleneyl (such as (C₆H₅)₂C═N, and the like), and silyl (such astert-butyldimethylsilyl, triisopropylsilyl, and the like), are known toone skilled in the art. The chemistry of amino protecting groups can befound in Wuts and Greene, Greene's Protective Groups in OrganicSynthesis, 4^(th) Ed., pp 696-926, John Wiley & Sons: New York, 2006. Insome embodiments, Pg¹ can be alkoxycarbonyl (such astert-butoxycarbonyl).

The amino protecting groups described above can be conveniently removedusing many available amino deprotecting agents that are specific to thevarious groups mentioned above without affecting other desired portionsof the compound. The tert-butoxycarbonyl group can be removed (e.g.,hydrolyzed) from the nitrogen atom, for example, by treatment with anacid (such as trifluoroacetic acid, toluenesulfonic acid, hydrochloricacid, and the like); a combination of reagents (e.g., mixture of acetylchloride and methanol) known to generate an acid; or a Lewis acid (e.g.,BF₃.Et₂O). The benzyloxycarbonyl group can be removed (e.g.,hydrogenolyzed) from the nitrogen atom, for example, by treatment withhydrogen and a catalyst (such as palladium on carbon). In someembodiments, the amino deprotecting agent can be trifluoroacetic acid.In some embodiments, the amino deprotecting agent containstrifluoroacetic acid and >0.5% by volume of water, e.g., >1.0% by volumeof water, >1.5% by volume of water, >2.0% by volume of water, from about2% to about 10% by volume of water, from about 10% to about 20% byvolume of water, or from about 20% to about 50% by volume of water. Insome embodiments, the amino deprotecting agent can be a mixture oftrifluoroacetic acid and water in a volumetric ratio of about 98:2. Insome embodiments, the amino deprotecting agent can be hydrochloric acid,optionally in a solvent (such as water, THF, or dioxane). In suchembodiments, the hydrochloric acid can be present in a concentration ofabout 4 N, e.g., about 1 N, about 2 N, about 3 N, about 5 N, about 6 N,about 7 N, about 8 N, about 9 N, or about 10 N. In some embodiments, thedeprotection can be performed in an alcohol (such as isopropanol). Insome embodiments, the Step M (Scheme 1) can be performed at atemperature from about −10° C. to about 60° C., e.g., from about −10° C.to about 0° C., from about 0° C. to about 25° C., from about 25° C. toabout 45° C., or from about 45° C. to about 60° C.

A base can be used for the conversion (e.g., hydrolysis) of theoxadiazolone ring in F14 to reveal the amidoxime in F15, optionally in asolvent (Step N, Scheme 1). The protection of the amidoxime as theoxadiazolone can be useful to prevent adverse reactions of the hydroxylgroup or that of the amidoxime as a whole. The base can be either anorganic base such as an acyclic amine (e.g., triethylamine,diisopropylethylamine (DIPEA), etc.) or a cyclic amine (e.g.,pyrrolidine, piperidine, etc.); or an inorganic base such as alkali(e.g., NaOH, LiOH, KOH, Mg(OH)₂, etc.). The base can be made availablein the form of a resin (such as Amberlite® and the like). In somefurther embodiments, the base can be provided in the form of a solutionin water such as about 2N solution (e.g., about 0.5N solution, about 1Nsolution, about 1.5N solution, about 2.5N solution, from about 3N toabout 5N solution, from about 5N to about 10N solution). In someembodiments, the base is an alkali metal hydroxide (such as, sodiumhydroxide). In some embodiments, the base can be 2N NaOH solution inwater. In some embodiments, the solvent can be methanol ortetrahydrofuran (THF). In some embodiments, the Step N (Scheme 1) can beperformed at a temperature from about −10° C. to about 60° C., e.g.,from about −10° C. to about 0° C., from about 0° C. to about 25° C.,from about 25° C. to about 45° C., or from about 45° C. to about 60° C.

In Step L (Scheme 1), the compound of Formula F13 can be obtained bytreating a compound of Formula F12, or a salt thereof, withPg¹-NH-sulfonyl chloride, optionally in a solvent, followed by treatmentof the resulting mixture with an organic base to afford the compound ofFormula F13. This Step L (Scheme 1) transforms a primary amine F12 to asulfonyl urea F13 using a protected amino-sulfonyl chloride(Pg¹-NH—SO₂Cl). The protected amino-sulfonyl chloride can be preparedand immediately used in the reaction with F12. The protecting groupcould be selected from any of the protecting groups known in the art forprotecting amines or sulfonamides (supra). In some embodiments, Pg¹ canbe an alkoxycarbonyl group (such as tert-butoxycarbonyl). In suchembodiments, the alkoxycarbonyl NH-sulfonyl chloride can be obtained bythe reaction of an alcohol (such as, ethanol, tert-butyl alcohol and thelike) with chlorosulfonyl isocyanate (ClS(O)₂NCO). Appropriate solventsfor this reaction include, but are not limited to, halogenated solventssuch as dichloromethane and the like. The organic base can be any basethat serves to neutralize the HCl generated during the reaction of theprimary amine such as F12 and the protected amino-sulfonyl chloride. Theorganic base can include acyclic tertiary amines such astri(C₁₋₆)alkylamine (e.g., triethylamine, diisopropylethylamine (DIPEA)and the like), cyclic tertiary amines (e.g., N-methyl piperidine,1,4-diazabicyclo[2.2.2]octane (DABCO) and the like). In someembodiments, the organic base can be triethylamine. In some embodiments,this step can be performed at a temperature from about −10° C. to about60° C., e.g., from about −10° C. to about 0° C., from about 0° C. toabout 25° C., from about 25° C. to about 45° C., or from about 45° C. toabout 60° C.

Organic compounds can be reduced to a lower oxidizing state by usingreducing agents. Reduction usually involves addition of hydrogen atomsor removal of oxygen atoms from a group. Organic azides such as F11 canbe reduced to amines such as F12 (Step K, Scheme 1) by the addition ofhydrogen, either in the form of elemental hydrogen or using a hydridereagent (such as NaBH₄, LiAlH₄ and the like); using triphenylphosphine;or using a combination of sodium iodide, chlorotrimethylsilane, andmethanol. In some embodiments, the compound of Formula F12 can beobtained by reducing a compound of Formula F11, or a salt thereof. Insome embodiments, the reducing can be carried out in the presence ofsodium iodide, chlorotrimethylsilane, and methanol. In some embodiments,the molar ratio of sodium iodide and chlorotrimethylsilane can be about1.0, e.g., about 0.9, about 0.95, about 1.0, about 1.05, or about 1.1.In some embodiments, chlorotrimethylsilane can be added to the mixtureof F11, sodium iodide and methanol as a solution in methanol. In someembodiments, Step K (Scheme 1) can be performed at about roomtemperature, e.g., from about 10° C. to about 50° C., from about 15° C.to about 40° C., from about 20° C. to about 30° C., or from about 25° C.to about 30° C.

The amino compounds F12, in some cases, may prove challenging to obtainin substantially pure form as determined by HPLC or NMR spectroscopy andthe like. While not intending to be bound by theory, it is believed thatsome of these amines might be difficult to purify by silica gelchromatography due to increased high affinity to silica gel or due tounwanted degradation during purification. In such embodiments, referringnow to Scheme 2, the compound of Formula F12 can be purified by reactingthe compound of Formula F12 with an amino protecting agent to afford acompound of Formula F12′, or a salt thereof, wherein Pg²N is a protectedamine. This protection (Step K′) can be followed by purifying thecompound of Formula F12′ to provide a purified compound of Formula F12′and reacting the purified compound of Formula F12′ with an aminodeprotecting agent (Step K″) to provide a purified compound of FormulaF12. Amino protecting agents and amino deprotecting agents are known toone skilled in the art, such as those in Wuts and Greene (ibid). In someembodiments, the amino protecting agent is di-t-butyl dicarbonate(Boe20). In such embodiments, Pg²N is tert-butoxy carbonyl-NH. In suchembodiments, the amino deprotecting agent is a reagent capable ofremoving the Boc protecting group (supra). In such embodiments, theamino deprotecting agent is an acid (e.g., hydrochloric acid,trifluoroacetic acid and the like), optionally in a solvent (such aswater, THF, or dioxane). In some embodiments, the hydrochloric acid canbe present in a concentration of about 4N, e.g., about 1N, about 2N,about 3N, about 5N, about 6N, about 7N, about 8N, about 9N, or about10N. In some embodiments, the deprotection can be performed in analcohol (such as isopropanol). In some embodiments, step K′ or K″ can beperformed at a temperature from about −10° C. to about 60° C., e.g.,from about −10° C. to about 0° C., from about 0° C. to about 25° C.,from about 25° C. to about 45° C., or from about 45° C. to about 60° C.Appropriate purification methods are known to one skilled in the art andcan include chromatography, crystallization, sublimation and the like.In some embodiments, purification can be performed by chromatography onsilica gel. The purity of the compounds, in general, are determined byphysical methods such as measuring the melting point (in case of asolid), obtaining a NMR spectrum, or performing a HPLC separation. Ifthe melting point decreases, if unwanted signals in the NMR spectrum aredecreased, or if extraneous peaks in an HPLC trace are removed, thecompound can be said to have been purified. In some embodiments, thecompounds are substantially purified.

In some embodiments, the compound of Formula F11 (Scheme 1) can beobtained by treating a compound of Formula F10, or a salt thereof,wherein L¹ can be selected from alkylsulfonyl (such as methanesulfonyl),haloalkylsulfonyl (such as trifluoromethanesulfonyl), arylsulfonyl (suchas toluenesulfonyl) and the like; with an azide reagent to afford thecompound of Formula F11 (Step J). In some embodiments, L¹ isalkylsulfonyl. Azide reagents include any reagent capable of producing anucleophilic azide ion. Examples of azide reagents include alkali metalazides (such as sodium azide, potassium azide, etc.). In some optionalembodiments, the azide reagent such as sodium azide can be used incombination with sodium iodide. Appropriate solvents for thistransformation are polar solvents including DMF, DMSO, NMP and the like.In some embodiments, Step J can be carried out in DMF. Step J can becarried out at an elevated temperature e.g., from about 40° C. to about100° C., from about 50° C. to about 90° C., or from about 60° C. toabout 80° C. In some embodiments, Step J can be carried out at about 50°C. In some embodiments, Step J can be carried out at about 85° C.

The compound of Formula F10, or a salt thereof, can be obtained in asequence of steps shown in Scheme 3. The preparation of theintermediate, 4-amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide F2,has been described in J. Heterocycl. Chem. (1965), 2, 253, which isincorporated herein by reference in its entirety, and its conversion tothe chloro oxime F3 has been described in Synth. Commun. (1988), 18,1427, which is incorporated herein by reference in its entirety. Amines(such as primary or secondary amines including amines that containprotected functionalities, e.g., ethyl amine, 2-methoxyethylamine ordimethylamine) can be coupled to the chloro oxime F3, optionally in asolvent (such as ethyl acetate), followed by addition of an organic base(such as triethylamine or DIPEA to quench the HCl generated in thereaction) to provide amidoxime compounds F4. Rearrangement of thecompounds such as F4, to transpose the amino group on the ring carbonand the amino group on the oxime carbon, to provide compounds F5 can beachieved by the treatment of F4 with a base (such as KOH, NaOH, LiOH,Mg(OH)₂, Al(OH)₃ and the like), optionally in a solvent (such as water,ethanol, ethylene glycol and the like), and refluxing the reactionmixture at elevated temperature e.g., about 70° C., about 80° C., about90° C., about 100° C., about 110° C., about 120° C., about 130° C.,about 140° C., about 150° C., about 160° C., about 170° C., about 180°C., about 190° C., or about 200° C. The amidoxime F5 can again beactivated as a chloro oxime F6 by the addition of F5 to an aqueousacidic mixture containing hydrochloric acid, optionally including aceticacid. In this process for the conversion of F5 to F6, the acidic mixtureof F5 can be heated to a temperature of about 45° C., such as about 30°C., about 40° C., about 50° C., or about 60° C. to achieve dissolution.Sodium chloride can be added to this solution and then treated with anitrite reagent, which can optionally be provided as an aqueoussolution, at a temperature below about 0° C., such as below about −10°C., below about −5° C., below about 5° C., or below about 10° C. Thenitrite reagent is one capable of providing a nitrite anion. Nitritereagents include alkali metal nitrite (e.g., sodium nitrite, potassiumnitrite and the like) and organo nitrites (e.g., tetraethylammoniumnitrite) which includes an organic cation. In some embodiments, ethylacetate, THF or dioxane can be used as a co-solvent. The chloro oxime F6can be coupled with aromatic amines such as anilines, optionally in apolar solvent (such as methanol, water, ethanol and the like) atelevated temperatures such as about 50° C., about 60° C., about 70° C.,about 80° C., about 90° C., about 100° C., about 110° C., or about 120°C., optionally in the presence of an inorganic base (such as KHCO₃,NaHCO₃) to provide arylamidoxime F7. In some embodiments, the inorganicbase can be provided in the form of an aqueous solution. In someembodiments, the inorganic base can be added to the reaction mixture atan elevated temperature. The amidoxime functionality of F7 can then beprotected as an oxadiazolone using 1,1′-carbonyl diimidazole (CDI) in asolvent (such as ethyl acetate, dioxane, THF and the like) at elevatedtemperatures such as about 50° C., about 60° C., about 70° C., about 80°C., about 90° C., or about 100° C. The methoxy group of F8 can then beconverted to a hydroxyl group in F9 using a methoxy deprotecting agentknown to one skilled in the art, such as those in Wuts and Greene,Greene's Protective Groups in Organic Synthesis, 4^(th) Ed., pp 24-30,John Wiley & Sons: New York, 2006. For example, by addition of borontribromide to a cold (such as from about −78° C. to about 25° C., e.g.,from about −78° C. to about 10° C., from about −78° C. to about 0° C.,from about −78° C. to about −10° C., from about 0° C. to about 25° C.,or from about 0° C. to about 10° C.) solution of F8, optionally in asolvent such as a halogenated solvent (e.g., DCM, chloroform and thelike) or ethyl acetate. The primary hydroxyl group in F9 cansubsequently be activated as a leaving group L¹O— (see F10) bysequential treatment with L¹Cl, optionally in a solvent (such as ethylacetate or DCM), and an organic base to mop up the generated HCl (suchas triethylamine or DIPEA). L¹, for example, can be selected fromalkylsulfonyl (e.g., methanesulfonyl), haloalkylsulfonyl (e.g.,trifluoromethanesulfonyl), arylsulfonyl (e.g., toluenesulfonyl) and thelike. The compound F10 can then be treated with any nucleophile fordisplacement (such as by S_(N)2 mechanism) of the leaving group L¹O.

Alternately, the compound of Formula F12 can be obtained through asequence of steps depicted in Scheme 4.

Referring now to Scheme 4, in some embodiments, the compound of FormulaF12 can be obtained by reacting a compound of Formula F24, or a saltthereof, wherein Pg³N is a protected amine (e.g., (C₆H₅)₃C—NH,(C₆H₅)₂C═N and the like); with an amino deprotecting agent to afford thecompound of Formula F12. Treatment of a compound F24 to replace Pg³Nwith NH₂ (Step Q) can be accomplished by methods for the deprotection ofparticular amine protecting groups known to one skilled in the art, suchas those in Wuts and Greene, Greene's Protective Groups in OrganicSynthesis, 4^(th) Ed., pp 696-926, John Wiley & Sons: New York, 2006. Insome embodiments, when the Pg³N is (C₆H₅)₂C═N, the deprotecting agentcan be: an acid such as an organic acid (e.g., trifluoroacetic acid,methanesulfonic acid and the like) or an inorganic acid (e.g.,hydrochloric acid); hydrogen and palladium; or acidic hydroxylamine(NH₂OH). In some embodiments, when the Pg³N is (C₆H₅)₃C—NH, thedeprotecting agent can include an organic acid (such as trifluoroaceticacid methanesulfonic acid and the like) and optionally an organosilane;hydrogen and palladium; or sodium in liquid ammonia. Organosilanes arecompounds that contain at least one Si—H bond and the rest of the groupsattached to silicon are alkyl, aryl or a combination thereof. Examplesof organosilanes include trialkylsilane (e.g., tri(isopropyl)silane)),triarylsilane (e.g., triphenylsilane) or diphenylmethylsilane. The stepQ can be performed at a temperature from about −10° C. to about 60° C.,e.g., from about −10° C. to about 0° C., from about 0° C. to about 25°C., from about 25° C. to about 45° C., or from about 45° C. to about 60°C.

Compounds F24 which are protected secondary amines can be prepared bythe Mitsunobu reaction of alcohols F25 with protected primary amines F22in the presence of a coupling reagent (Step P). The coupling reagent canbe a combination of a tertiary phosphine such as triarylphosphine (e.g.,triphenylphosphine) or trialkylphosphine (e.g., tributylphosphine) and adialkyl azodicarboxylate. Dialkyl azodicarboxylates possess a generalstructure: ROOC—N═N—COOR, where R can be an alkyl group (e.g.,diisopropyl azodicarboxylate, diethyl azodicarboxylate, ordi-p-chlorobenzyl azodicarboxylate). While not intending to be bound bytheory, it is believed that amine protection with trifluoroacetyl moiety(such as in F22) prevents side reactions and improves the yield of thesecondary amine F24. The hydroxyl group of alcohols such as F25 can beactivated in the presence of the coupling reagent. The amine nucleophilecan displace the activated hydroxyl group to form the secondary amine.The Mitsunobu reaction can be performed in a solvent such as an ethere.g., THF, dioxane, dialkyl ether and the like; halogenated solventse.g., dichloromethane, chloroform and the like; non-polar solvents e.g.,benzene, toluene and the like; polar-aprotic solvents such as DMF, HMPAand the like. In some embodiments, the compound of Formula F24 can beobtained by treating a compound of Formula F22, or a salt thereof, witha compound of Formula F25, or a salt thereof, and a coupling reagent toprovide the compound of Formula F24. In some embodiments, this step canbe performed at a temperature from about −10° C. to about 60° C., e.g.,from about −10° C. to about 0° C., from about 0° C. to about 25° C.,from about 25° C. to about 45° C., or from about 45° C. to about 60° C.

Compounds F22 can be made by a two step process (Steps G′ and O) fromcompounds F21. Compounds F21 can be treated with 1,1′-carbonyldiimidazole (CDI), optionally in a solvent (such as ethyl acetate orTHF), at an elevated temperature such as about 50° C., e.g., about 60°C., about 65° C., about 70° C., about 80° C., or about 90° C., toconvert the amidoxime in compounds F21 to oxadiazolone present incompounds F26. These compounds F26 in turn can be treated withtrifluoroacetic anhydride, optionally in a solvent (such as DCM, THF,dioxane, or ethyl acetate) in the presence of an organic base (such aspyridine, triethylamine, DIPEA and the like) to provide compounds F22.In some embodiments, the compound of Formula F22 can be obtained bytreating a compound of Formula F21, or a salt thereof, with carbonyldiimidazole (CDI) to afford a compound of Formula F26, or a saltthereof, and treating the compound of Formula F26 with trifluoroaceticanhydride to afford the compound of Formula F22.

Referring now to Scheme 5 (Step P′) and based on the above descriptionof Mitsunobu reaction, another aspect of the invention provides aprocess for preparing a compound of Formula F8, or a salt thereof,wherein, R², R³, and n are defined herein; including reacting a compoundof Formula F22, or a salt thereof, and a compound of Formula F25′, or asalt thereof, with a coupling reagent, optionally in a solvent (such asTHF, dialkyl ether, or dichloromethane), to provide the compound ofFormula F8. In some embodiments, this step can be performed at atemperature from about −10° C. to about 60° C., e.g., from about −10° C.to about 0° C., from about 0° C. to about 25° C., from about 25° C. toabout 45° C., or from about 45° C. to about 60° C.

Scheme 6 delineates an alternative route for the introduction of thesulfonamide group to the amino compound F12. Treatment of F12 withsulfamide in the presence of a base (Step R) such as an organic basewhich can be a heterocyclic base (e.g., pyridine), or a trialkylamine(e.g., triethylamine, DIPEA and the like), each of which can optionallybe used as a solvent for this transformation, can provide sulfonyl ureassuch as F14. This reaction can be carried out at elevated temperaturessuch as about 130° C., e.g., about 100° C., about 110° C., about 120°C., about 130° C., or about 140° C. Such heating can be favorablyapplied using microwave irradiation. Microwave irradiation can beperformed in a commercial microwave oven (e.g., the Initiator™,available from Biotage) operating in a single mode fashion. CompoundsF14 containing the oxadiazolone ring can be deprotected (e.g.,hydrolyzed) to the desired amidoximes F15 in the presence of a base(Step N′). The base can be either an organic base such as an acyclicamine (e.g., triethylamine, diisopropylethylamine (DIPEA), etc.) or acyclic amine (e.g., pyrrolidine, piperidine, etc.); or an inorganic basesuch as alkali (e.g., NaOH, LiOH, KOH, Mg(OH)₂, etc.). The base can bemade available in the form of a resin (such as Amberlite® and the like).In some further embodiments, the base can be provided in the form of asolution in water such as about 2N solution (e.g., about 0.5N solution,about 1N solution, about 1.5N solution, about 2.5N solution, from about3N to about 5N solution, from about 5N to about 10N solution). In someembodiments, the base can be an alkali metal hydroxide (such as, sodiumhydroxide). In some embodiments, the base can be a 2N NaOH solution inwater. In some embodiments, the solvent can be methanol ortetrahydrofuran (THF). In some embodiments, the deprotection can beperformed at a temperature from about −10° C. to about 60° C., e.g.,from about −10° C. to about 0° C., from about 0° C. to about 25° C.,from about 25° C. to about 45° C., or from about 45° C. to about 60° C.Hence, this aspect of the invention provides a process for preparing acompound of Formula F15, or a salt thereof, wherein R², R³, and n, areas defined herein; including reacting a compound of Formula F12, or asalt thereof, with sulfamide and an organic base to afford a compound ofFormula F14, or a salt thereof, and reacting the compound of FormulaF14, or a salt thereof, with a base to afford the compound of FormulaF15.

The present invention further provides a compound of Formula F9, F12,and F14, or a salt thereof, wherein R² is Cl, Br, CF₃, CH₃, or CN; R³ isH or F; and n is 1 or 2.

In some embodiments, R² is Br, R³ is F, and n is 1.

In some embodiments, R² is Br, R³ is F, and n is 2.

In some embodiments, R² is Cl, R³ is F, and n is 1.

In some embodiments, R² is Cl, R³ is F, and n is 2.

In some embodiments, R² is CF₃, R³ is F, and n is 1.

In some embodiments, R² is CF₃, R³ is F, and n is 2.

In some embodiments, R² is CF₃, R³ is H, and n is 1.

In some embodiments, R² is CF₃, R³ is H, and n is 2.

In some embodiments, R² is CH₃, R³ is F, and n is 1.

In some embodiments, R² is CN, R³ is F, and n is 1.

Referring now to Scheme 7, compounds F19 can be obtained from primaryamino compounds F12 by treatment with methanesulfonyl chloride (Step S),optionally in a solvent such as ethyl acetate, halogenated solvents(e.g., dichloromethane, chloroform and the like) or ethereal solvents(THF, diethyl ether, dioxane and the like), in the presence of anorganic base (to mop up the generated HCl) such as tri(C₁₋₆)alkylamine(e.g., triethylamine, DIPEA and the like), or pyridine to affordsulfonamides F20. The methanesulfonyl group can be replaced with otheralkylsulfonyl (e.g., ethylsulfonyl), haloalkylsulfonyl (e.g.,trifluoromethanesulfonyl), arylsulfonyl (e.g., toluenesulfonyl) and thelike, without altering the procedures. In some embodiments, this stepcan be performed at a temperature from about −10° C. to about 60° C.,e.g., from about −10° C. to about 0° C., from about 0° C. to about 25°C., from about 25° C. to about 45° C., or from about 45° C. to about 60°C. The sulfonamide compounds F20 containing the oxadiazolone ring can bedeprotected (e.g., hydrolyzed) to the desired amidoximes F19 in thepresence of a base (Step N″). The base can be either an organic basesuch as an acyclic amine (e.g., triethylamine, diisopropylethylamine(DIPEA), etc.) or a cyclic amine (e.g., pyrrolidine, piperidine, etc.);or an inorganic base such as alkali metal hydroxide or alkaline earthmetal hydroxide (e.g., NaOH, LiOH, KOH, Mg(OH)₂, etc.). The base can bemade available in the form of a resin (such as Amberlite® and the like).In some further embodiments, the base can be provided in the form of asolution in water such as about 2N solution (e.g., about 0.5N solution,about 1N solution, about 1.5N solution, about 2.5N solution, from about3N to about 5N solution, from about 5N to about 10N solution). In someembodiments, the base is an alkali metal hydroxide (e.g., sodiumhydroxide). In some embodiments, the base can be 2N NaOH solution inwater. In some embodiments, the solvent can be methanol ortetrahydrofuran (THF). In some embodiments, the deprotection can beperformed at a temperature from about −10° C. to about 60° C., e.g.,from about −10° C. to about 0° C., from about 0° C. to about 25° C.,from about 25° C. to about 45° C., or from about 45° C. to about 60° C.Accordingly, another aspect of the invention provides a process forpreparing a compound of Formula F19, or a salt thereof, wherein R², R³,and n, are as defined herein; including reacting a compound of FormulaF12, or a salt thereof, with methanesulfonyl chloride in the presence ofan organic base to afford a compound of Formula F20, or a salt thereof,and reacting the compound of Formula F20 with a base to afford thecompound of Formula F19. In some embodiments, the base can be an alkalimetal hydroxide such as sodium hydroxide (e.g., 2N NaOH).

Aryl or alkylsulfonamides (e.g., methanesulfonamides F19) can beobtained by the sequence of steps shown in Scheme 8. Mono-protectedl,n-diamines such as F40 (e.g., commercially availableN-(aminoalkyl)(t-butoxy)carboxamide) can be treated with sulfonylchlorides such as arylsulfonyl chlorides or alkylsulfonyl chlorides(e.g., methanesulfonyl chloride), optionally in a solvent such as ethylacetate, halogenated solvents (e.g., dichloromethane, chloroform and thelike) or ethereal solvents (THF, diethyl ether, dioxane and the like),in the presence of an organic base (to mop up the generated HCl) such astriethylamine, pyridine, DIPEA and the like, to provide sulfonamides F41(Step S′). The protecting group on mono-protected l,n-diamines F40 maybe selected from the various amino protecting groups and a suitabledeprotection conditions can be appropriately selected (supra) to affordamine F33 (Step M′). In some embodiments, protecting group can bealkoxycarbonyl (such as tert-butoxycarbonyl, Boc). In such embodiments,the amino deprotecting agent can be an acid e.g., hydrochloric acid ortrifluoroacetic acid, optionally in a solvent (such as dioxane).

The preparation of chloro oxime F3 has been described in Synth. Commun.(1988), 18, 1427, which is incorporated herein by reference in itsentirety. Amines (such as primary or secondary amines including aminesthat contain protected functionalities, e.g., ethyl amine,2-methoxyethylamine, dimethylamine or F33) can be coupled to the chlorooxime F3, optionally in a solvent (such as ethyl acetate or ethanol),followed by addition of an organic base (such as triethylamine or DIPEAto quench the HCl generated in the reaction) to provide amidoximecompounds F16 (Step C′). In some embodiments, this step can be performedat a temperature from about −10° C. to about 60° C., e.g., from about−10° C. to about 0° C., from about 0° C. to about 25° C., from about 25°C. to about 45° C., or from about 45° C. to about 60° C. Rearrangementof the compounds such as F16 to transpose the amino group on the ringcarbon and the amino group on the oxime carbon to provide compounds suchas F17 (Step D′) can be achieved by the treatment of F16 with a base(such as KOH, NaOH, LiOH, Mg(OH)₂, Al(OH)₃ and the like), optionally ina solvent (such as water, ethanol, ethylene glycol and the like), andrefluxing the reaction mixture at elevated temperature e.g., about 70°C., about 80° C., about 90° C., about 100° C., about 110° C., about 120°C., about 130° C., about 140° C., about 150° C., about 160° C., about170° C., about 180° C., about 190° C., or about 200° C. The amidoximeF17 can again be activated as a chloro oxime F18 by the addition of F17to an aqueous acidic mixture containing hydrochloric acid, optionallyincluding acetic acid (Step E′). In this process for the conversion ofF17 to F18, the acidic mixture of F17 can be heated to temperature about45° C., such as about 30° C., about 40° C., about 50° C., or about 60°C. to achieve dissolution. Sodium chloride can be added to this solutionand treated with a nitrite reagent, which can optionally be provided asan aqueous solution, at a temperature below about 0° C. such as belowabout −10° C., below about −5° C., below about 5° C., or below about 10°C. The nitrite reagent is one capable of providing a nitrite anion.Nitrite reagents include alkali metal nitrite (e.g., sodium nitrite,potassium nitrite and the like) and organo nitrites (e.g.,tetraethylammonium nitrite) which includes an organic cation. In someembodiments, ethyl acetate, THF or dioxane can be used as a co-solvent.The substitution of the chloride in F18 with aromatic amines such asanilines F27, optionally in a polar solvent (such as methanol, water,ethanol and the like), at room temperature can affordmethanesulfonamides F19 (Step F′). In some embodiments, temperaturessuch as about 10° C., about 20° C., about 30° C., about 40° C., or about50° C. can be employed. This reaction can be optionally carried out inthe presence of an inorganic base (such as KHCO₃, NaHCO₃) which can beprovided in the form of an aqueous solution.

Accordingly, in another aspect of the invention provides a process forpreparing a compound of Formula F19, or a salt thereof, wherein R², R³,and n, are as defined herein; including reacting a compound of FormulaF17, or a salt thereof, with hydrochloric acid, optionally in a solvent(such as dioxane), followed by treatment with a nitrite reagent (suchas, sodium nitrite), optionally in the form of an aqueous solution, toafford a compound of Formula F18, or a salt thereof, and reacting thecompound of Formula F18 with a compound of Formula F27, or a saltthereof, to afford the compound of Formula F19.

In some embodiments, the compound of Formula F17 can be obtained bytreating a compound of Formula F16, or a salt thereof, with a base (suchas potassium hydroxide) in a solvent (such as ethylene glycol) at atemperature sufficient to reflux the solvent (such as 130° C.), toprovide a compound of Formula F17.

The present invention further provides a compound of Formula F18, or asalt thereof, wherein n is 1 or 2. In some embodiments, n is 1. In someembodiments, n is 2.

Compounds F28 can be obtained as described in Scheme 9. The chloro oximeF6 (supra, Scheme 1) can be coupled with heterocyclic amines (such ascompound of Formula F38), optionally in a polar solvent (such asmethanol, water, ethanol and the like), in the presence of a base suchas an inorganic base or an organic base (e.g., Et₃N, pyridine or DIPEA)to provide arylamidoxime F36 (Step F″). In some embodiments, theconversion of F6 to F36 can be carried out at temperatures such as about10° C., about 20° C., about 30° C., about 40° C., about 50° C., about60° C., or about 90° C. In some embodiments, the inorganic base can beprovided in the form of an aqueous solution. In some embodiments, theinorganic base can be added to the reaction mixture at an elevatedtemperature. The amidoxime functionality of F36 can then be protected asan oxadiazolone using 1,1′-carbonyl diimidazole (CDI) in a solvent (suchas ethyl acetate, dioxane, THF and the like) at elevated temperaturessuch as about 50° C., about 60° C., about 70° C., about 80° C., about90° C., or about 100° C. (Step G″). The methoxy group of F35 can then beconverted to a hydroxyl group in F34 by methods known to one skilled inthe art for the deprotecetion of methoxy group (Step H′), such as thosein Wuts and Greene, Greene's Protective Groups in Organic Synthesis,4^(th) Ed., pp 24-30, John Wiley & Sons: New York, 2006. For example, byaddition of boron tribromide to a cold (such as from about −78° C. toabout 25° C., e.g., from about −78° C. to about 10° C., from about −78°C. to about 0° C., from about −78° C. to about −10° C., from about 0° C.to about 25° C., or from about 0° C. to about 10° C.) solution of F35,optionally in a solvent such as a halogenated solvent (e.g., DCM,chloroform and the like) or ethyl acetate. The primary hydroxyl group inF34 can then be subsequently activated as a leaving group L¹O— (see StepI′, F32) by sequential treatment with L¹Cl, optionally in a solvent(such as ethyl acetate or DCM), and an organic base to mop up thegenerated HCl (such as triethylamine or DIPEA). In compounds F32, L¹ canbe selected from alkylsulfonyl (e.g., methanesulfonyl),haloalkylsulfonyl (e.g., trifluoromethanesulfonyl), arylsulfonyl (e.g.,toluenesulfonyl) and the like. The compound F32 can then be treated withany nucleophile for S_(N)2 displacement of the leaving group L¹O. Insome embodiments, this step can be performed at a temperature from about−10° C. to about 60° C., e.g., from about −10° C. to about 0° C., fromabout 0° C. to about 25° C., from about 25° C. to about 45° C., or fromabout 45° C. to about 60° C.

When the nucleophile is an azide ion, F32 provides F31 (Step J′). Azidereagents include any reagent capable of producing a nucleophilic azideion. Examples of azide reagents include alkali metal azides (such assodium azide, potassium azide). In some optional embodiments, the azidereagent such as sodium azide can be used in combination with sodiumiodide. Appropriate solvents for this transformation are polar solventsincluding DMF, DMSO, NMP and the like. In some embodiments, this stepcan be carried out in DMF. In some embodiments, this step can be carriedout at an elevated temperature e.g., from about 40° C. to about 100° C.,from about 50° C. to about 90° C., or from about 60° C. to about 80° C.In some embodiments, this step can be carried out at 50° C. In someembodiments, this step can be carried out at 85° C. Organic azides suchas F31 can be reduced to organic amines such as F29 by the addition ofhydrogen, either in the form of elemental hydrogen; using a hydridereagent (such as NaBH₄, LiAlH₄ and the like); using triphenylphosphine;or using a combination of sodium iodide, chlorotrimethylsilane, andmethanol (Step K′″). In some embodiments, the reducing can be carriedout in the presence of sodium iodide, chlorotrimethylsilane, andmethanol. In some embodiments, the reduction can be performed at aboutroom temperature e.g., from about 10° C. to about 50° C., from about 15°C. to about 40° C., from about 20° C. to about 30° C., or from about 25°C. to about 30° C. In some embodiments, the molar ratio of sodium iodideand chlorotrimethylsilane can be about 1.0 e.g., about 0.9, about 0.95,about 1.0, about 1.05, or about 1.1. In some embodiments,chlorotrimethylsilane can be added to the mixture of F31, sodium iodideand methanol as a solution in methanol.

Treatment of F29 with sulfamide in the presence of a base such as anorganic base which can be a heterocyclic base (e.g., pyridine), or atrialkylamine (e.g., triethylamine, DIPEA and the like), each of whichcan optionally be used as a solvent for this transformation to providethe sulfonyl ureas such as F30 (Step R′). This reaction can be carriedout at elevated temperatures such as about 130° C., e.g., about 100° C.,about 110° C., about 120° C., about 130° C., or about 140° C. Suchheating can be favorably applied using microwave irradiation. Microwaveirradiation can be performed in a commercial microwave oven (e.g., theInitiator™, available from Biotage) operating in a single mode fashion.Compounds F30 containing the oxadiazolone ring can be deprotected (e.g.,hydrolyzed) to the desired amidoximes F28 in the presence of a base(Step N′″). The base can be either an organic base such as acyclicamines (e.g., triethylamine, diisopropylethylamine (DIPEA), etc.) orcyclic amines (e.g., pyrrolidine, piperidine, etc.); or an inorganicbase such as alkali (e.g., NaOH, LiOH, KOH, Mg(OH)₂, etc.). The base canbe made available in the form of a resin (such as Amberlite® and thelike). In some further embodiments, the base can be provided in the formof a solution in water (an aqueous base) such as about 2N solution(e.g., about 0.5N solution, about 1N solution, about 1.5N solution,about 2.5N solution, from about 3N to about 5N solution, from about 5Nto about 10N solution). In some embodiments, the base can be an alkalimetal hydroxide (e.g., sodium hydroxide). In some embodiments, the basecan be a 2N NaOH solution in water. In some embodiments, the solvent canbe methanol or tetrahydrofuran (THF). In some embodiments, thedeprotection can be performed at a temperature from about −10° C. toabout 60° C., e.g., from about −10° C. to about 0° C., from about 0° C.to about 25° C., from about 25° C. to about 45° C., or from about 45° C.to about 60° C.

Accordingly, another aspect of the invention provides a process forpreparing a compound of Formula F28, or a salt thereof, wherein R⁴ is F,Cl, Br, or I; and n is 1 or 2; including reacting a compound of FormulaF29, or a salt thereof, with sulfamide and an organic base to afford acompound of Formula F30, or a salt thereof, and reacting the compound ofFormula F30 with a base to afford the compound of Formula F28.

In some embodiments, R⁴ is Cl and n is 1.

In some embodiments, R⁴ is Br and n is 1.

In some embodiments, reacting a compound of Formula F29 further includesheating the reaction (such as using microwave irradiation).

In another aspect, the invention provides a process of obtaining thecompound of Formula F29 by reducing a compound of Formula F31, or a saltthereof. In some embodiments, the reducing can be carried out with acombination of sodium iodide, chlorotrimethylsilane, and methanol.

In another aspect of the invention, the compound of Formula F31 can beobtained by treating a compound of Formula F32, or a salt thereof,wherein L¹ is selected from alkylsulfonyl, haloalkylsulfonyl, andarylsulfonyl; with an azide reagent to afford the compound of FormulaF31.

As used herein, the term “alkyl,” when used alone or together withadditional moiety terms, refers to a straight-chained or branched,saturated hydrocarbon group having from 1 to 6 carbon atoms, 1 to 4carbon atoms, or 1 to 3 carbon atoms. Example alkyl groups includemethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, andthe like.

As used herein, “alkenyl” refers to an alkyl group having one or moredouble carbon-carbon bonds. Example alkenyl groups include ethenyl(vinyl), propenyl, and the like.

As used herein, the term “aryl” refers to an aromatic hydrocarbon groupwhich can be mono- or polycyclic having from 6 to 14 carbon atoms.Example aryl groups include phenyl, naphthyl, anthracenyl,phenanthrenyl, indanyl, indenyl, and the like.

As used herein, the term “haloalkyl,” when used alone or together withan additional moiety, refers to an alkyl group substituted by one ormore halogen atoms independently selected from F, Cl, Br, and I. Examplehaloalkyl groups include CF₃, CHF₂, CH₂CF₃, and the like.

As used herein, the term “alkoxy” refers to an —O-alkyl group. Examplealkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy andisopropoxy), t-butoxy, and the like.

As used herein, “alkylamine” refers to an amino (NH₂) group substitutedby an alkyl group. Example alkylamine groups include methylamine,hexylamine, and the like.

As used herein, “trialkylamine” refers to a nitrogen atom substituted bythree alkyl group. Example trialkylamine groups include trimethylamine,triethylamine, and the like.

As used herein, the term “alkoxycarbonyl” refers to CO substituted by analkoxy group: —C(O)—O-alkyl. Example alkoxycarbonyl groups includeethoxycarbonyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),9-fluorenylmethyloxycarbonyl (Fmoc), and the like.

As used herein, the term “alkylsulfonyl” refers to a sulfonyl groupsubstituted by an alkyl group: alkylS(O)₂—. Example alkylsulfonyl groupsinclude, methanesulfonyl, ethanesulfonyl, and the like.

As used herein, the term “haloalkylsulfonyl” refers to a sulfonyl groupsubstituted by a haloalkyl group. Example haloalkylsulfonyl groupsinclude, trifluoromethanesulfonyl, 1,1,1-trifluoroethanesulfonyl, andthe like.

As used herein, the term “arylsulfonyl” refers to a sulfonyl groupsubstituted by an aryl group or a substituted aryl group, wherein thesubstituents on the aryl group are selected from halo, nitro, C₁₋₄alkyl, and C₁₋₄ haloalkyl.

As used herein, the term “heterocyclic base” refers to a 4 to 14membered, optionally substituted, heterocycle wherein at least one ringforming member is a nitrogen atom. The heterocyclic base can be aromaticor non-aromatic. Example heterocyclic bases include pyridine,pyrrolidine, piperidine, morpholine etc. Example substituents on theheterocycle include F, Cl, Br, C₁₋₄ alkyl, and C₁₋₄ haloalkyl.

Methods of Use

Compounds of the invention can inhibit activity of the enzymeindoleamine-2,3-dioxygenase (IDO). For example, the compounds of theinvention can be used to inhibit activity of IDO in cell or in anindividual in need of modulation of the enzyme by administering aninhibiting amount of a compound of the invention.

The present invention further provides methods of inhibiting thedegradation of tryptophan in a system containing cells expressing IDOsuch as a tissue, living organism, or cell culture. In some embodiments,the present invention provides methods of altering (e.g., increasing)extracellular tryptophan levels in a mammal by administering aneffective amount of a compound of composition provided herein. Methodsof measuring tryptophan levels and tryptophan degradation are routine inthe art.

The present invention further provides methods of inhibitingimmunosuppression such as IDO-mediated immunosuppression in a patient byadministering to the patient an effective amount of a compound orcomposition recited herein. IDO-mediated immunosuppression has beenassociated with, for example, cancers, tumor growth, metastasis, viralinfection, viral replication, etc.

The present invention further provides methods of treating diseasesassociated with activity or expression, including abnormal activityand/or overexpression, of IDO in an individual (e.g., patient) byadministering to the individual in need of such treatment atherapeutically effective amount or dose of a compound of the presentinvention or a pharmaceutical composition thereof. Example diseases caninclude any disease, disorder or condition that is directly orindirectly linked to expression or activity of the IDO enzyme, such asover expression or abnormal activity. An IDO-associated disease can alsoinclude any disease, disorder or condition that can be prevented,ameliorated, or cured by modulating enzyme activity. Examples ofIDO-associated diseases include cancer, viral infection such as HIVinfection, HCV infection, depression, neurodegenerative disorders suchas Alzheimer's disease and Huntington's disease, trauma, age-relatedcataracts, organ transplantation (e.g., organ transplant rejection), andautoimmune diseases including asthma, rheumatoid arthritis, multiplesclerosis, allergic inflammation, inflammatory bowel disease, psoriasisand systemic lupus erythematosusor. Example cancers treatable by themethods herein include cancer of the colon, pancreas, breast, prostate,lung, brain, ovary, cervix, testes, renal, head and neck, lymphoma,leukemia, melanoma, and the like. The compounds of the invention canalso be useful in the treatment of obesity and ischemia.

As used herein, the term “cell” is meant to refer to a cell that is invitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can bepart of a tissue sample excised from an organism such as a mammal. Insome embodiments, an in vitro cell can be a cell in a cell culture. Insome embodiments, an in vivo cell is a cell living in an organism suchas a mammal.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” the IDO enzyme with a compound of the inventionincludes the administration of a compound of the present invention to anindividual or patient, such as a human, having IDO, as well as, forexample, introducing a compound of the invention into a samplecontaining a cellular or purified preparation containing the IDO enzyme.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal, individualor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician.

As used herein the term “treating” or “treatment” refers to 1)preventing the disease; for example, preventing a disease, condition ordisorder in an individual who may be predisposed to the disease,condition or disorder but does not yet experience or display thepathology or symptomatology of the disease; 2) inhibiting the disease;for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder (i.e., arrestingfurther development of the pathology and/or symptomatology), or 3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology).

Combination Therapy

One or more additional pharmaceutical agents or treatment methods suchas, for example, anti-viral agents, chemotherapeutics or otheranti-cancer agents, immune enhancers, immunosuppressants, radiation,anti-tumor and anti-viral vaccines, cytokine therapy (e.g., IL2, GM-CSF,etc.), and/or tyrosine kinase inhibitors can be used in combination withthe compounds of the present invention for treatment of IDO-associateddiseases, disorders or conditions. The agents can be combined with thepresent compounds in a single dosage form, or the agents can beadministered simultaneously or sequentially as separate dosage forms.

Suitable antiviral agents contemplated for use in combination with thecompounds of the present invention can comprise nucleoside andnucleotide reverse transcriptase inhibitors (NRTIs), non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors and otherantiviral drugs.

Example suitable NRTIs include zidovudine (AZT); didanosine (ddl);zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir(1592U89); adefovir dipivoxil [bis(POM)-PMEA]; lobucavir (BMS-180194);BCH-10652; emitricitabine [(−)-FTC]; beta-L-FD4 (also called beta-L-D4Cand named beta-L-2′,3′-dicleoxy-5-fluoro-cytidene); DAPD,((−)-beta-D-2,6,-diamino-purine dioxolane); and lodenosine (FddA).Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine(BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442(1-(ethoxy-methyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione);and (+)-calanolide A (NSC-675451) and B. Typical suitable proteaseinhibitors include saquinavir (Ro 31-8959); ritonavir (ABT-538);indinavir (MK-639); nelfnavir (AG-1343); amprenavir (141 W94); lasinavir(BMS-234475); DMP-450; BMS-2322623; ABT-378; and AG-1 549. Otherantiviral agents include hydroxyurea, ribavirin, IL-2, IL-12,pentafuside and Yissum Project No. 11607.

Suitable chemotherapeutic or other anti-cancer agents include, forexample, alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes) such as uracil mustard, chlormethine, cyclophosphamide(Cytoxan™), ifosfamide, melphalan, chlorambucil, pipobroman,triethylene-melamine, triethylenethiophosphoramine, busulfan,carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

In the treatment of melanoma, suitable agents for use in combinationwith the compounds of the present invention include: dacarbazine (DTIC),optionally, along with other chemotherapy drugs such as carmustine(BCNU) and cisplatin; the “Dartmouth regimen,” which consists of DTIC,BCNU, cisplatin and tamoxifen; a combination of cisplatin, vinblastine,and DTIC; or temozolomide. Compounds according to the invention may alsobe combined with immunotherapy drugs, including cytokines such asinterferon alpha, interleukin 2, and tumor necrosis factor (TNF) in thetreatment of melanoma.

Compounds of the invention may also be used in combination with vaccinetherapy in the treatment of melanoma. Antimelanoma vaccines are, in someways, similar to the anti-virus vaccines which are used to preventdiseases caused by viruses such as polio, measles, and mumps. Weakenedmelanoma cells or parts of melanoma cells called antigens may beinjected into a patient to stimulate the body's immune system to destroymelanoma cells.

Melanomas that are confined to the arms or legs may also be treated witha combination of agents including one or more compounds of theinvention, using a hyperthermic isolated limb perfusion technique. Thistreatment protocol temporarily separates the circulation of the involvedlimb from the rest of the body and injects high doses of chemotherapyinto the artery feeding the limb, thus providing high doses to the areaof the tumor without exposing internal organs to these doses that mightotherwise cause severe side effects. Usually the fluid is warmed to 102°to 104° F. Melphalan is the drug most often used in this chemotherapyprocedure. This can be given with another agent called tumor necrosisfactor (TNF) (see section on cytokines).

Suitable chemotherapeutic or other anti-cancer agents include, forexample, antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors) such as methotrexate, 5-fluorouracil, floxuridine,cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,pentostatine, and gemcitabine.

Suitable chemotherapeutic or other anti-cancer agents further include,for example, certain natural products and their derivatives (forexample, vinca alkaloids, antitumor antibiotics, enzymes, lymphokinesand epipodophyllotoxins) such as vinblastine, vincristine, vindesine,bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin,idarubicin, ara-C, paclitaxel (TAXOL™), mithramycin, deoxycoformycin,mitomycin-C, L-asparaginase, interferons (especially IFN-α), etoposide,and teniposide.

Other cytotoxic agents include navelbene, CPT-11, anastrazole,letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, anddroloxafine.

Also suitable are cytotoxic agents such as epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes such as cis-platin andcarboplatin; biological response modifiers; growth inhibitors;antihormonal therapeutic agents; leucovorin; tegafur; and haematopoieticgrowth factors.

Other anti-cancer agent(s) include antibody therapeutics such astrastuzumab (Herceptin), antibodies to costimulatory molecules such asCTLA-4, 4-1BB and PD-1, or antibodies to cytokines (IL-10, TGF-β, etc.).

Other anti-cancer agents also include those that block immune cellmigration such as antagonists to chemokine receptors, including CCR2 andCCR4.

Other anti-cancer agents also include those that augment the immunesystem such as adjuvants or adoptive T cell transfer.

Anti-cancer vaccines include dendritic cells, synthetic peptides, DNAvaccines and recombinant viruses.

Methods for the safe and effective administration of most of thesechemotherapeutic agents are known to those skilled in the art. Inaddition, their administration is described in the standard literature.For example, the administration of many of the chemotherapeutic agentsis described in the “Physicians' Desk Reference” (PDR, e.g., 1996edition, Medical Economics Company, Montvale, N.J.), the disclosure ofwhich is incorporated herein by reference as if set forth in itsentirety.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of the invention can beadministered in the form of pharmaceutical compositions which is acombination of a compound of the invention and a pharmaceuticallyacceptable carrier. These compositions can be prepared in a manner wellknown in the pharmaceutical art, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration may be topical(including ophthalmic and to mucous membranes including intranasal,vaginal and rectal delivery), pulmonary (e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal), ocular, oral orparenteral. Methods for ocular delivery can include topicaladministration (eye drops), subconjunctival, periocular or intravitrealinjection or introduction by balloon catheter or ophthalmic insertssurgically placed in the conjunctival sac. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal, orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. Parenteral administration can be inthe form of a single bolus dose, or may be, for example, by a continuousperfusion pump. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of the inventionabove in combination with one or more pharmaceutically acceptablecarriers. In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 100 mg, more usually about 10 to about30 mg, of the active ingredient. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpre-formulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepre-formulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid pre-formulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention canvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. The proportion or concentration of a compound of theinvention in a pharmaceutical composition can vary depending upon anumber of factors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, thecompounds of the invention can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral administration. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The compounds of the invention can also be formulated in combinationwith one or more additional active ingredients which can include anypharmaceutical agent such as anti-viral agents, vaccines, antibodies,immune enhancers, immune suppressants, anti-inflammatory agents and thelike.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to fluorescent dye, spinlabel, heavy metal or radio-labeled compounds of the invention thatwould be useful not only in imaging but also in assays, both in vitroand in vivo, for localizing and quantitating the IDO enzyme in tissuesamples, including human, and for identifying IDO enzyme ligands byinhibition binding of a labeled compound. Accordingly, the presentinvention includes IDO enzyme assays that contain such labeledcompounds.

The present invention further includes isotopically-labeled compounds ofFormula I. An “isotopically” or “radio-labeled” compound is a compoundof the invention where one or more atoms are replaced or substituted byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature (i.e., naturallyoccurring). Suitable radionuclides that may be incorporated in compoundsof the present invention include but are not limited to ²H (also writtenas D for deuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C,¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I,¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that is incorporated in theinstant radio-labeled compounds will depend on the specific applicationof that radio-labeled compound. For example, for in vitro IDO enzymelabeling and competition assays, compounds that incorporate ³H, ¹⁴C,⁸²Br, ¹²⁵I, ¹³¹I or ³⁵S will generally be most useful. For radio-imagingapplications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br willgenerally be most useful.

It is understood that a “radio-labeled” or “labeled compound” is acompound that has incorporated at least one radionuclide. In someembodiments the radionuclide is selected from the group consisting of³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br.

Synthetic methods for incorporating radio-isotopes into organiccompounds are applicable to compounds of the invention and are wellknown in the art.

A radio-labeled compound of the invention can be used in a screeningassay to identify/evaluate compounds. In general terms, a newlysynthesized or identified compound (i.e., test compound) can beevaluated for its ability to reduce binding of the radio-labeledcompound of the invention to the IDO enzyme. Accordingly, the ability ofa test compound to compete with the radio-labeled compound for bindingto the IDO enzyme directly correlates to its binding affinity.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of IDO-associated diseases ordisorders, obesity, diabetes and other diseases referred to herein whichinclude one or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of theinvention. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc., as will be readily apparent tothose skilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results. The compounds of the Examples were found to be inhibitorsof IDO according to one or more of the assays provided herein.

EXAMPLES Example 14-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A: 4-Amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Malononitrile [Aldrich, product #M1407] (320.5 g, 5 mol) was added towater (7 L) preheated to 45° C. and stirred for 5 min. The resultingsolution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol)was added. When the temperature reached 10° C., 6 N hydrochloric acid(55 mL) was added. A mild exothermic reaction ensued with thetemperature reaching 16° C. After 15 min the cold bath was removed andthe reaction mixture was stirred for 1.5 hrs at 16-18° C. The reactionmixture was cooled to 13° C. and 50% aqueous hydroxylamine (990 g, 15mol) was added all at once. The temperature rose to 26° C. When theexothermic reaction subsided the cold bath was removed and stirring wascontinued for 1 hr at 26-27° C., then it was slowly brought to reflux.Reflux was maintained for 2 hrs and then the reaction mixture wasallowed to cool overnight. The reaction mixture was stirred in an icebath and 6 N hydrochloric acid (800 mL) was added in portions over 40min to pH 7.0. Stirring was continued in the ice bath at 5° C. Theprecipitate was collected by filtration, washed well with water anddried in a vacuum oven (50° C.) to give the desired product (644 g,90%). LCMS for C₃H₆N₅O₂ (M+H)⁺: m/z=144.0. ¹³C NMR (75 MHz, CD₃OD): δ156.0, 145.9, 141.3.

Step B: 4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride

4-Amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol)was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Nhydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at42-45° C. until complete solution was achieved. Sodium chloride (518 g,3 eq.) was added and this solution was stirred in an ice/water/methanolbath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL)was added over 3.5 hrs while maintaining the temperature below 0° C.After complete addition stirring was continued in the ice bath for 1.5hrs and then the reaction mixture was allowed to warm to 15° C. Theprecipitate was collected by filtration, washed well with water, takenin ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g)and stirred for 1 hr. This suspension was filtered through sodiumsulfate (200 g) and the filtrate was concentrated on a rotaryevaporator. The residue was dissolved in methyl t-butyl ether (5.5 L),treated with charcoal (40 g), stirred for 40 min and filtered throughCelite. The solvent was removed in a rotary evaporator and the resultingproduct was dried in a vacuum oven (45° C.) to give the desired product(256 g, 53.4%). LCMS for C₃H₄ClN₄O₂ (M+H)⁺: m/z=162.9. ¹³C NMR (100 MHz,CD₃OD): δ 155.8, 143.4, 129.7.

Step C:4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g,1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5° C.2-methoxyethylamine [Aldrich, product #143693] (119.0 mL, 1.35 mol) wasadded in one portion while stirring. The reaction temperature rose to41° C. The reaction was cooled to 0-5° C. Triethylamine (258 mL, 1.84mol) was added. After stirring 5 min, LCMS indicated reactioncompletion. The reaction solution was washed with water (500 mL) andbrine (500 mL), dried over sodium sulfate, and concentrated to give thedesired product (294 g, 119%) as a crude dark oil. LCMS for C₆H₁₂N₅O₃(M+H)⁺: m/z=202.3. ¹H NMR (400 MHz, DMSO-d₆): δ 10.65 (s, 1H), 6.27 (s,2H), 6.10 (t, J=6.5 Hz, 1H), 3.50 (m, 2H), 3.35 (d, J=5.8 Hz, 2H), 3.08(s, 3H).

Step D:N′-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide(248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210g, 3.7 mol) was added. The reaction was refluxed at 100° C. overnight(15 hours). TLC with 50% ethyl acetate (containing 1% ammoniumhydroxide) in hexane indicated reaction completed (product Rf=0.6,starting material Rf=0.5). LCMS also indicated reaction completion. Thereaction was cooled to room temperature and extracted with ethyl acetate(3×1 L). The combined ethyl acetate solution was dried over sodiumsulfate and concentrated to give the desired product (201 g, 81%) as acrude off-white solid. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z=202.3 ¹H NMR (400MHz, DMSO-d₆): δ 10.54 (s, 1H), 6.22 (s, 2H), 6.15 (t, J=5.8 Hz, 1H),3.45 (t, J=5.3 Hz, 2H), 3.35 (m, 2H), 3.22 (s, 3H).

Step E:N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride

At room temperatureN′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueoussolution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) wasadded followed by water (250 mL) and ethyl acetate (250 mL). At 3-5° C.a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at3-8° C. for 2 hours and then room temperature over the weekend. LCMSindicated reaction completed. The reaction solution was extracted withethyl acetate (2×200 mL). The combined ethyl acetate solution was driedover sodium sulfate and concentrated to give the desired product (49.9g, 126%) as a crude white solid. LCMS for C₆H₁₀ClN₄O₃ (M+H)⁺: m/z=221.0.¹H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1H), 5.85 (t, J=5.6 Hz, 1H), 3.50(t, J=5.6 Hz, 2H), 3.37 (dd, J=10.8, 5.6 Hz, 2H), 3.25 (s, 3H).

Step F:N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixturewas heated to 60° C. 3-Bromo-4-fluoroaniline [Oakwood products, product#013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warmsodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was addedover 15 min. The reaction was stirred at 60° C. for 20 min. LCMSindicated reaction completion. The reaction solution was cooled to roomtemperature and extracted with ethyl acetate (2×300 mL). The combinedethyl acetate solution was dried over sodium sulfate and concentrated togive the desired product (76.7 g, 98%) as a crude brown solid. LCMS forC₁₂H₁₄BrFN₅O₃ (M+H)⁺: m/z=374.0, 376.0. ¹H NMR (400 MHz, DMSO-d₆): δ11.55 (s, 1H), 8.85 (s, 1H), 7.16 (t, J=8.8 Hz, 1H), 7.08 (dd, J=6.1,2.7 Hz, 1H), 6.75 (m, 1H), 6.14 (t, J=5.8 Hz, 1H), 3.48 (t, J=5.2 Hz,2H), 3.35 (dd, J=10.8, 5.6 Hz, 2H), 3.22 (s, 3H).

Step G:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A mixture ofN-(3-bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(76.5 g, 0.204 mol), 1,1′-carbonyldiimidazole (49.7 g, 0.307 mol), andethyl acetate (720 mL) was heated to 60° C. and stirred for 20 min. LCMSindicated reaction completed. The reaction was cooled to roomtemperature, washed with 1 N HCl(2×750 mL), dried over sodium sulfate,and concentrated to give the desired product (80.4 g, 98%) as a crudebrown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z=400.0, 402.0. ¹H NMR(400 MHz, DMSO-d₆): δ 7.94 (t, J=8.2 Hz, 1H), 7.72 (dd, J=9.1, 2.3 Hz,1H), 7.42 (m, 1H), 6.42 (t, J=5.7 Hz, 1H), 3.46 (t, J=5.4 Hz, 2H), 3.36(t, J=5.8 Hz, 2H), 3.26 (s, 3H).

Step H:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At −67°C. boron tribromide (37 mL, 0.392 mol) was added over 15 min. Thereaction was warmed up to −10° C. in 30 min. LCMS indicated reactioncompleted. The reaction was stirred at room temperature for 1 hour. At0-5° C. the reaction was slowly quenched with saturated sodiumbicarbonate solution (1.5 L) over 30 min. The reaction temperature roseto 25° C. The reaction was extracted with ethyl acetate (2×500 mL, firstextraction organic layer is on the bottom and second extraction organiclager is on the top). The combined organic layers were dried over sodiumsulfate and concentrated to give the desired product (75 g, 99%) as acrude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z=386.0, 388.0. ¹HNMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.70 (m, 1H),7.68 (t, J=8.7 Hz, 1H), 6.33 (t, J=5.6 Hz, 1H), 4.85 (t, J=5.0 Hz, 1H),3.56 (dd, J=10.6, 5.6 Hz, 2H), 3.29 (dd, J=11.5, 5.9 Hz, 2H).

Step I:2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate

To a solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(1.5 kg, 3.9 mol, containing also some of the correspondingbromo-compound) in ethyl acetate (12 L) was added methanesulfonylchloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature.Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, duringwhich time the reaction temperature increased to 35° C. After 2 h, thereaction mixture was washed with water (5 L), brine (1 L), dried oversodium sulfate, combined with 3 more reactions of the same size, and thesolvents removed in vacuo to afford the desired product (7600 g,quantitative yield) as a tan solid. LCMS for C₁₃H₁₁BrFN₅O₆SNa (M+Na)⁺:m/z=485.9, 487.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz,1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75 (t, J=5.9 Hz, 1H), 4.36(t, J=5.3 Hz, 2H), 3.58 (dd, J=11.2, 5.6 Hz, 2H), 3.18 (s, 3H).

Step J:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

To a solution of2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate (2.13 kg, 4.6 mol, containing also some of thecorresponding bromo-compound) in dimethylformamide (4 L) stirring in a22 L flask was added sodium azide (380 g, 5.84 mol). The reaction washeated at 50° C. for 6 h, poured into ice/water (8 L), and extractedwith 1:1 ethyl acetate:heptane (20 L). The organic layer was washed withwater (5 L) and brine (5 L), and the solvents removed in vacuo to affordthe desired product (1464 g, 77%) as a tan solid. LCMS forC₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z=433.0, 435.0. ¹H NMR (400 MHz, DMSO-d₆): δ8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75(t, J=5.7 Hz, 1H), 3.54 (t, J=5.3 Hz, 2H), 3.45 (dd, J=11.1, 5.2 Hz,2H).

Step K:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

Sodium iodide (1080 g, 7.2 mol) was added to3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for30 min during which time a mild exotherm was observed.Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution inmethanol (1 L) dropwise at a rate so that the temperature did not exceed35° C., and the reaction was allowed to stir for 3.5 h at ambienttemperature. The reaction was neutralized with 33 wt % solution ofsodium thiosulfate pentahydrate in water (˜1.5 L), diluted with water (4L), and the pH adjusted to 9 carefully with solid potassium carbonate(250 g—added in small portions: watch foaming). Di-tert-butyldicarbonate (318 g, 1.45 mol) was added and the reaction was allowed tostir at room temperature. Additional potassium carbonate (200 g) wasadded in 50 g portions over 4 h to ensure that the pH was still at orabove 9. After stirring at room temperature overnight, the solid wasfiltered, triturated with water (2 L), and then MTBE (1.5 L). A total of11 runs were performed (5.5 kg, 13.38 mol). The combined solids weretriturated with 1:1 THF:dichloromethane (24 L, 4 runs in a 20 L rotaryevaporator flask, 50° C., 1 h), filtered, and washed withdichloromethane (3 L each run) to afford an off-white solid. The crudematerial was dissolved at 55° C. tetrahydrofuran (5 mL/g), treated withdecolorizing carbon (2 wt %) and silica gel (2 wt %), and filtered hotthrough celite to afford the product as an off-white solid (5122 g). Thecombined MTBE, THF, and dichloromethane filtrates were concentrated invacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethylacetate gradient, 30 L) to afford more product (262 g). The combinedsolids were dried to a constant weight in a convection oven (5385 g,83%).

In a 22 L flask was charged hydrogen chloride (4 N solution in1,4-dioxane, 4 L, 16 mol).tert-Butyl[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate(2315 g, 4.77 mol) was added as a solid in portions over 10 min. Theslurry was stirred at room temperature and gradually became a thickpaste that could not be stirred. After sitting overnight at roomtemperature, the paste was slurried in ethyl acetate (10 L), filtered,re-slurried in ethyl acetate (5 L), filtered, and dried to a constantweight to afford the desired product as a white solid (combined withother runs, 5 kg starting material charged, 4113 g, 95%). LCMS forC₁₂H₁₁BrFN₆O₃ (M+H)⁺: m/z=384.9, 386.9. ¹H NMR (400 MHz, DMSO-d₆): δ8.12 (m, 4H), 7.76 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.78 (t, J=6.1 Hz,1H), 3.51 (dd, J=11.8, 6.1 Hz, 2H), 3.02 (m, 2H).

Step L:tert-Butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate[Aldrich, product #142662] (149 mL, 1.72 mol) and dichloromethane (1.5L) and cooled using an ice bath to 2° C. tert-Butanol (162 mL, 1.73 mol)in dichloromethane (200 mL) was added dropwise at a rate so that thetemperature did not exceed 10° C. The resulting solution was stirred atroom temperature for 30-60 min to providetert-butyl[chlorosulfonyl]carbamate.

A 22 L flask was charged with3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After coolingto −15° C. with an ice/salt bath, the solution oftert-butyl[chlorosulfonyl]carbamate (prepared as above) was added at arate so that the temperature did not exceed −10° C. (addition time 7min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) wasadded at a rate so that the temperature did not exceed −5° C. (additiontime 10 min). The cold bath was removed, the reaction was allowed towarm to 10° C., split into two portions, and neutralized with 10% concHCl (4.5 L each portion). Each portion was transferred to a 50 Lseparatory funnel and diluted with ethyl acetate to completely dissolvethe white solid (˜25 L). The layers were separated, and the organiclayer was washed with water (5 L), brine (5 L), and the solvents removedin vacuo to afford an off-white solid. The solid was triturated withMTBE (2×1.5 L) and dried to a constant weight to afford a white solid. Atotal of 4113 g starting material was processed in this manner (5409 g,98%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.90 (s, 1H), 8.08 (dd, J=6.2, 2.5Hz, 1H), 7.72 (m, 1H), 7.59 (t, J=8.6 Hz, 1H), 6.58 (t, J=5.7 Hz, 1H),3.38 (dd, J=12.7, 6.2 Hz, 2H), 3.10 (dd, J=12.1, 5.9 Hz, 2H), 1.41 (s,9H).

Step M:N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) wasaddedtert-butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate(1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixturewas stirred at room temperature for 1.5 h, the solvents removed invacuo, and chased with dichloromethane (2 L). The resulting solid wastreated a second time with fresh 98:2 trifluoroacetic acid:water (8.9L), heated for 1 h at 40-50° C., the solvents removed in vacuo, andchased with dichloromethane (3×2 L). The resulting white solid was driedin a vacuum drying oven at 50° C. overnight. A total of 5409 g wasprocessed in this manner (4990 g, quant. yield). LCMS for C₁₂H₁₂BrFN₇O₅S(M+H)⁺: m/z=463.9, 465.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2,2.5 Hz, 1H), 7.72 (m, 1H), 7.59 (t, J=8.7 Hz, 1H), 6.67 (t, J=5.9 Hz,1H), 6.52 (t, J=6.0 Hz, 1H), 3.38 (dd, J=12.7, 6.3 Hz, 2H), 3.11 (dd,J=12.3, 6.3 Hz).

Step N:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

To a crude mixture ofN-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(2.4 mol) containing residual amounts of trifluoroacetic acid stirringin a 22 L flask was added THF (5 L). The resulting solution was cooledto 0° C. using an ice bath and 2 N NaOH (4 L) was added at a rate sothat the temperature did not exceed 10° C. After stirring at ambienttemperature for 3 h (LCMS indicated no starting material remained), thepH was adjusted to 3-4 with concentrated HCl (˜500 mL). The THF wasremoved in vacuo, and the resulting mixture was extracted with ethylacetate (15 L). The organic layer was washed with water (5 L), brine (5L), and the solvents removed in vacuo to afford a solid. The solid wastriturated with MTBE (2×2 L), combined with three other reactions of thesame size, and dried overnight in a convection oven to afford a whitesolid (3535 g). The solid was recrystallized (3×22 L flasks, 2:1water:ethanol, 14.1 L each flask) and dried in a 50° C. convection ovento a constant weight to furnish the title compound as an off-white solid(3290 g, 78%). LCMS for C₁₁H₁₄BrFN₇O₄S (M+H)⁺: m/z=437.9, 439.9. ¹H NMR(400 MHz, DMSO-d₆): δ 11.51 (s, 1H), 8.90 (s, 1H), 7.17 (t, J=8.8 Hz,1H), 7.11 (dd, J=6.1, 2.7 Hz, 1H), 6.76 (m, 1H), 6.71 (t, J=6.0 Hz, 1H),6.59 (s, 2H), 6.23 (t, J=6.1 Hz, 1H), 3.35 (dd, J=10.9, 7.0 Hz, 2H),3.10 (dd, J=12.1, 6.2 Hz, 2H).

The final product was an anhydrous crystalline solid. The water contentwas determined to be less than 0.1% by Karl Fischer titration. The X-raypowder diffraction (XRPD) pattern was determined (Rigaku MiniFlex PowderDiffractometer; Cu at 1.054056 Å with Kβ filter; start angle=3, stopangle=45, sampling=0.02, scan speed=2) and is shown in FIG. 1. A list of2-theta peaks is provided in Table 1 below. The melting range of thesolid was determined on a Mettler Toledo Differential ScanningCaloimetry (DSC) 822 instrument. The sample was heated from 40° C. to240° C. at a heating rate of 10° C. per min. The DSC thermogram (FIG. 2)showed a T_(onset) at 162.7° C. and T_(peak) at 163.8° C.Thermogravimetric analysis (TGA) (FIG. 3) showed weight loss of 0.3%,heating from 20° C. to 150° C. at a heating rate of 10° C./min using aTA Instrument Q500.

TABLE 1 2-Theta Height H % 3.9 74 1.1 7.2 119 1.8 13.4 180 2.8 14.0 1502.3 15.9 85 1.3 18.4 903 13.9 18.9 1469 22.7 21.3 519 8 21.8 6472 10022.7 516 8 23.9 2515 38.9 24.8 804 12.4 25.3 182 2.8 27.4 476 7.4 28.6354 5.5 29.2 1767 27.3 29.9 266 4.1 30.6 773 11.9 31.2 379 5.8 31.6 2914.5 32.7 144 2.2 33.5 221 3.4 36.4 469 7.2 37.6 152 2.3 38.7 1381 21.341.0 153 2.4 42.1 382 5.9 43.6 527 8.1 44.4 1080 16.7

Example 2N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 17step E, usingN′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-bromo-4-fluoroaniline [Oakwood Products, Inc., product #013091] asthe starting materials. LCMS for C₁₂H₁₅BrFN₆O₄S (M+H)⁺: m/z=437.0,439.0. ¹H NMR (400 MHz, DMSO-d₆): δ 11.49 (s, 1H), 8.90 (s, 1H), 7.17(m, 2H), 7.09 (dd, J=6.3, 2.5 Hz, 1H), 6.26 (t, J=6.1 Hz, 1H), 3.33 (m,2H), 3.13 (q, J=6.0 Hz, 2H), 2.89 (s, 3H).

Example 34-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A:3-(4-Amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example5, step A, using4-amino-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide[see U. S. Pat. App. Pub. No. 2006/0258719] as the starting material in98% yield. LCMS for C₁₀H₆BrFN₅O₃ (M+H)⁺: m/z=342.0, 344.0. ¹H NMR (400MHz, DMSO-d₆): δ 8.06 (dd, J=6.2, 2.5 Hz, 1H), 7.72-7.67 (m, 1H), 7.58(dd, J=8.7, 8.7 Hz, 1H), 6.60 (s, 2H).

Step B:N-{4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide

The desired compound was prepared according to the procedure of Example5, step B, using3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-oneas the starting material in 81% yield. LCMS for C₁₂H₅BrF₄N₅O₄ (M+H)⁺:m/z=437.9, 439.9. ¹H NMR (400 MHz, DMSO-d₆): δ 7.92-7.89 (m, 1H),7.54-7.52 (m, 2H).

Step C:4-(3-Bromo-4-fluorophenyl)-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A solution of 3-methoxypropan-1-ol [Fluka product #38457] (3.1 mL, 32mmol) and triphenylphosphine (8.4 g, 32 mmol) in tetrahydrofuran (93 mL)at 0° C. was treated with diisopropyl azodicarboxylate (6.7 mL, 34 mmol)dropwise. The reaction mixture was stirred at 0° C. for 15 min, treatedwith a solution ofN-{4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide(10 g, 23 mmol) in tetrahydrofuran (47 mL), and stirred at 25° C. for 72h. The reaction mixture was concentrated, diluted with ethyl acetate(200 mL), treated with trifluoroacetic acid (20 mL) and water (20 mL),and heated at 50° C. for 6 h. The reaction mixture was concentrated,rediluted with ethyl acetate (200 mL) and washed with water (3×80 mL),saturated sodium bicarbonate (2×80 mL) and brine (80 mL), dried overanhydrous sodium sulfate, filtered, and concentrated to a crude residue.This material was purified on silica gel to give the desired product(6.4 g, 54%) as a white solid. LCMS for C₁₄H₁₄BrFN₅O₄ (M+H)⁺: m/z=414.0,416.0.

Step D:4-(3-Bromo-4-fluorophenyl)-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(6.3 g, 14 mmol) in dichloromethane (60 mL) at −78° C. was treated with1 M boron tribromide in dichloromethane (28 mL, 28 mmol) and stirred at25° C. for 2 h. The reaction mixture was cooled to 0° C. and quenchedwith saturated sodium bicarbonate (100 mL). The aqueous layer wasseparated and extracted with dichloromethane (2×150 mL). The combinedorganic layers were washed with brine (100 mL), dried over anhydroussodium sulfate, filtered, and concentrated to a crude off-white solid.This material was purified on silica gel to give the desired product(4.0 g, 73%) as a white solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z=400.0,402.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.07 (dd, J=6.2, 2.5 Hz, 1H),7.72-7.68 (m, 1H), 7.59 (dd, J=8.8, 8.6 Hz, 1H), 6.54 (t, J=5.7 Hz, 1H),4.60 (t, J=5.1 Hz, 1H), 3.48-3.43 (m, 2H), 3.32-3.26 (m, 2H), 1.74-1.67(m, 2H).

Step E:3-{4-[(3-Azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

A solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(3.0 g, 7.5 mmol) in dichloromethane (27 mL) was treated withmethanesulfonyl chloride (0.75 mL, 9.7 mmol) andN,N-diisopropylethylamine (2.6 mL, 15 mmol) and stirred at 25° C. for 2h. The reaction mixture was diluted with water (20 mL) and extractedwith dichloromethane (20 mL). The organic layer was separated, driedover anhydrous sodium sulfate, filtered, and concentrated to give themesylate which was used without further purification. A solution of thecrude mesylate in N,N-dimethylformamide (24 mL) was treated with sodiumazide (0.73 g, 11 mmol) and heated at 85° C. for 2 h. The reactionmixture was diluted with ethyl acetate (300 mL) and washed with water(100 mL), saturated sodium bicarbonate (100 mL), and brine (100 mL),dried over anhydrous sodium sulfate, filtered, and concentrated to givethe desired product (3.2 g, 99%). This material was used without furtherpurification. LCMS for C₁₃H₁₀BrFN₈O₃Na (M+Na)⁺: m/z=446.9, 448.9.

Step F:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide

A solution of3-{4-[(3-azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(2.0 g, 4.7 mmol) in methanol (36 mL) was treated with sodium iodide(4.2 g, 28 mmol) and stirred at 25° C. for 5 min. The reaction mixturewas treated with a solution of chlorotrimethylsilane (3.6 mL, 28 mmol)in methanol (7 mL) dropwise and stirred at 25° C. for 40 min. Thereaction mixture was slowly poured into a solution of sodium thiosulfate(5.0 g, 32 mmol) in water (200 mL) that was cooled at 0° C. The solidthat precipitated was filtered, washed with water, and dried to give thedesired product (2.3 g, 93%) as a solid. LCMS for C₁₃H₁₃BrFN₆O₃ (M+H)⁺:m/z=399.0, 401.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.1, 2.3 Hz,1H), 7.74-7.70 (m, 1H), 7.60 (dd, J=8.8, 8.6 Hz, 1H), 7.22 (br s, 2H),6.69 (br s, 1H), 2.81-2.77 (m, 2H), 1.86-1.79 (m, 2H).

Step G:N-[3-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)propyl]sulfamide

A solution of3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide (150 mg, 0.28 mmol) and sulfamide (160 mg, 1.7 mmol) inpyridine (2.5 mL) was heated in a microwave at 130° C. for 10 min. Thereaction mixture was concentrated to give a crude residue. This materialwas purified by preparative LCMS to give the desired product (96 mg,71%) as a solid. LCMS for C₁₃H₁₄BrFN₇O₅S (M+H)⁺: m/z=478.0, 480.0. ¹HNMR (400 MHz, DMSO-d₆): δ 8.07 (dd, J=6.2, 2.5 Hz, 1H), 7.73-7.69 (m,1H), 7.59 (dd, J=8.8, 8.6 Hz, 1H), 6.57-6.51 (m, 4H), 3.31-3.26 (m, 2H),2.92-2.87 (m, 2H), 1.79-1.72 (m, 2H).

Step H:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

A solution ofN-[3-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)propyl]sulfamide(35 mg, 73 μmol) in methanol (1 mL) was treated with 2 M NaOH (0.3 mL,0.6 mmol) and stirred at 25° C. for 30 min. The reaction mixture wastreated with acetic acid (50 μL, 0.9 mmol), filtered, and purified bypreparative LCMS to give the desired product (14 mg, 42%) as a solid.LCMS for C₁₂H₁₆BrFN₇O₄S (M+H)⁺: m/z=451.8, 453.9. ¹H NMR (400 MHz,DMSO-d₆): δ 11.5 (s, 1H), 8.89 (s, 1H), 7.17 (dd, J=8.8, 8.6 Hz, 1H),7.09 (dd, J=6.1, 2.7 Hz, 1H), 6.76-6.72 (m, 1H), 6.56 (dd, J=6.1, 6.1Hz, 1H), 6.51 (s, 2H), 6.17 (dd, J=5.9, 5.9 Hz, 1H), 3.27-3.21 (m, 2H),2.94-2.88 (m, 2H), 1.78-1.71 (m, 2H).

Example 4N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

Step A: tert-Butyl {3-[(methylsulfonyl)amino]propyl}carbamate

The desired compound was prepared according to the procedure of Example17, step A, using N-(3-aminopropyl)(tert-butoxy)carboxamide [Aldrichproduct #436992] as the starting material in 70% yield. LCMS forC₄H₁₃N₂O₂S ([M−Boc+H]+H)⁺: m/z=153.1.

Step B: N-(3-Aminopropyl)methanesulfonamide hydrochloride

The desired compound was prepared according to the procedure of Example17, step B, using tert-butyl {3-[(methylsulfonyl)amino]propyl}carbamateas the starting material. LCMS for C₄H₁₃N₂O₂S (M+H)⁺: m/z=153.1.

Step C:4-Amino-N′-hydroxy-N-{3-[(methylsulfonyl)amino]propyl}-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example17, step C, using N-(3-aminopropyl)methanesulfonamide hydrochloride and4-amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride [madeaccording to Example 1, steps A through B] as the starting materials in19% yield.

Step D:N′-Hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example17, step D, using4-amino-N′-hydroxy-N-{3-[(methylsulfonyl)amino]propyl}-1,2,5-oxadiazole-3-carboximidamideas the starting material. LCMS for C₇H₁₅N₆O₄S (M+H)⁺: m/z=279.0.

Step E:N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example17, step E, usingN′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-bromo-4-fluoroaniline [Oakwood Products, Inc., product #013091] asthe starting materials in 12% yield. LCMS for C₁₃H₁₇BrFN₆O₄S (M+H)⁺:m/z=451.0, 453.0. ¹H NMR (400 MHz, CD₃OD): δ 7.12 (dd, J=5.9, 2.4 Hz,1H), 7.05 (t, J=8.7 Hz, 1H), 6.83 (m, 1H), 3.39 (t, J=6.8 Hz, 2H), 3.14(t, J=6.6 Hz, 2H), 2.94 (s, 3H), 1.87 (m, 2H).

Example 54-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A:3-(4-Amino-1,2,5-oxadiazol-3-yl)-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

A solution of4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide(80 g, 0.29 mol) [see US Pat. App. Pub. No. 2006/0258719] intetrahydrofuran (500 mL) was treated with a solution of1,1′-carbonyldiimidazole (53 g, 0.32 mol) in tetrahydrofuran (200 mL)and heated at reflux for 1 h. The reaction mixture was cooled to 25° C.and concentrated to the point where a large amount of solidprecipitated. The heterogeneous mixture was diluted with ethyl acetate(1.5 L) and washed with 1 N HCl (2×300 mL), water (300 mL), and brine(200 mL). The organic layer was separated, dried over anhydrous sodiumsulfate, filtered, and concentrated to give the desired product (88 g,quantitative) as an off-white solid. This material was used withoutfurther purification. LCMS for C₁₀H₆ClFN₅O₃ (M+H)⁺: m/z=298.0. ¹H NMR(400 MHz, DMSO-d₆): δ 7.96 (dd, J=6.6, 2.3 Hz, 1H), 7.69-7.60 (m, 2H),6.60 (s, 2H).

Step B:N-{4-[4-(3-Chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide

A solution of3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(15 g, 50 mmol) in dichloromethane (120 mL) was treated withtrifluoroacetic anhydride (14 mL, 100 mmol), cooled to 0° C., andtreated with pyridine (8.2 mL, 100 mmol). The reaction mixture wasstirred at 25° C. for 10 min, cooled to 0° C., and quenched with water(10 mL). The reaction mixture was diluted with ethyl acetate (500 mL)and washed with 1 N HCl (300 mL), water (2×200 mL), and brine (200 mL).The organic layer was separated, dried over anhydrous sodium sulfate,filtered, and concentrated to ˜50 mL volume. This solution was warmed(˜40-50° C.) and treated with hexanes (600 mL) under vigorous stirring,followed by petroleum ether (200 mL). The mixture was stirred at 0° C.for 30 min and the solid was collected by filtration, washed withhexanes, and dried to give the desired product (19.7 g, 99%) as a whitesolid. LCMS for C₁₂H₅ClF₄N₅O₄ (M+H)⁺: m/z=394.0. ¹H NMR (400 MHz,DMSO-d₆): δ 7.82 (dd, J=6.6, 2.5 Hz, 1H), 7.59 (dd, J=9.0, 9.0 Hz, 1H),7.52-7.47 (m, 1H).

Step C:4-(3-Chloro-4-fluorophenyl)-3-(4-{[2-(tritylamino)ethyl]amino}-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one

A solution of 2-(tritylamino)ethanol (10 g, 33 mmol) [EP599220 and J.Org. Chem. (2001), 66, 7615] and triphenylphosphine (8.7 g, 33 mmol) intetrahydrofuran (65 mL) at 0° C. was treated with diisopropylazodicarboxylate (7.0 mL, 35 mmol) dropwise. The reaction mixture wasstirred at 0° C. for 15 min, treated with a solution ofN-{4-[4-(3-chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide(9.3 g, 24 mmol) in tetrahydrofuran (28 mL), and stirred at 25° C. for16 h. The reaction mixture was concentrated, diluted with ethyl acetate(350 mL), cooled to 0° C., treated with 1 N HCl (200 mL), and stirred at25° C. for 1 h. The reaction mixture was treated with additional 1 N HCl(150 mL) and stirred at 25° C. for 3 h. The organic layer was separated,washed with saturated sodium bicarbonate (200 mL) and brine (100 mL),dried over anhydrous sodium sulfate, filtered, and concentrated to ayellow foam which was reconcentrated from hexanes to give an oily solid.The oily solid was treated with methyl tert-butyl ether (50 mL) andstirred to give a heterogeneous mixture. The solid was filtered, washedwith methyl tert-butyl ether (30 mL), and dried to give the desiredproduct (10 g, 74%) as a white solid. LCMS for C₃₁H₂₄ClFN₆O₃Na (M+Na)⁺:m/z=605.2. ¹H NMR (300 MHz, DMSO-d₆): δ 7.97 (dd, J=6.7, 2.6 Hz, 1H),7.71-7.66 (m, 1H), 7.60 (dd, J=9.1, 8.8 Hz, 1H), 7.40-7.37 (m, 6H),7.28-7.23 (m, 6H), 7.18-7.12 (m, 3H), 6.59 (dd, J=5.9, 5.6 Hz, 1H),3.37-3.31 (m, 2H), 2.96 (dd, J=7.6, 7.6 Hz, 1H), 2.27-2.19 (m, 2H).

Step D:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

A premixed solution of triisopropylsilane (3.4 mL, 17 mmol) andtrifluoroacetic acid (44 mL, 570 mmol) was added to4-(3-chloro-4-fluorophenyl)-3-(4-{[2-(tritylamino)ethyl]amino}-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one(6.5 g, 11 mmol) and the resulting suspension was stirred at 25° C. for30 min. The reaction mixture was filtered and washed withtrifluoroacetic acid. The filtrate was concentrated to an oil which wasdiluted with methanol (25 mL), cooled to 0° C., treated with 4 M HCl in1,4-dioxane (14 mL), and stirred at 25° C. for 15 min. The mixture wasconcentrated to a solid that was treated with diethyl ether (50 mL) andfiltered. The solid was washed with diethyl ether (50 mL) and dried togive the desired product (4.1 g, 98%) as a white solid. LCMS forC₁₂H₁₁ClFN₆O₃ (M+H)⁺: m/z=341.1. ¹H NMR (300 MHz, DMSO-d₆): δ 8.05-8.00(m, 4H), 7.75-7.69 (m, 1H), 7.64 (dd, J=9.1, 8.8 Hz, 1H), 6.77 (dd,J=5.9, 5.9 Hz, 1H), 3.54-3.47 (m, 2H), 3.04-2.99 (m, 2H).

Step E:N-[2-({4-[4-(3-Chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

A solution of chlorosulfonyl isocyanate (2.0 mL, 23 mmol) indichloromethane (70 mL) was treated with t-butyl alcohol (2.2 mL, 23mmol) at 0° C. and stirred at 25° C. for 1 h. This mixture was added toa suspension of3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride (4.3 g, 11 mmol) in dichloromethane (70 mL). The reactionmixture was treated with a solution of triethylamine (6.3 mL, 45 mmol)in dichloromethane (20 mL) at 0° C. and stirred at 25° C. for 3 h. Thereaction mixture was diluted with 0.1 N HCl and extracted with ethylacetate (2×100 mL). The combined organic layers were washed with brine(100 mL), dried over anhydrous sodium sulfate, filtered, andconcentrated to a white solid. The white solid was diluted withdichloromethane (100 mL), treated with trifluoroacetic acid (20 mL), andstirred at 25° C. for 3 h. The reaction mixture was concentrated to acrude residue that was purified by silica gel chromatography to give thedesired product (3.7 g, 78%) as a white solid. LCMS for C₁₂H₁₂ClFN₇O₅S(M+H)⁺: m/z=420.0. ¹H NMR (300 MHz, DMSO-d₆): δ 7.98 (dd, J=6.4, 2.1 Hz,1H), 7.70-7.60 (m, 2H), 6.66 (t, J=5.9 Hz, 1H), 6.57 (s, 2H), 6.52 (t,J=5.9 Hz, 1H), 3.42-3.35 (m, 2H), 3.13-3.06 (m, 2H).

Step F:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

A solution ofN-[2-({4-[4-(3-chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(3.7 g, 8.8 mmol) in methanol (70 mL) was treated with 2 M NaOH (18 mL,35 mmol) and stirred at 25° C. for 2 h. The reaction mixture wasquenched with 6 N HCl to pH-7 and the methanol was removed under reducedpressure.

The solid that precipitated was filtered and washed with water to givethe desired product (3.2 g, 92%) as a white solid. LCMS forC₁₁H₁₄ClFN₇O₄S (M+H)⁺: m/z=394.0. ¹H NMR (400 MHz, DMSO-d₆): δ 7.96 (dd,J=6.8, 2.1 Hz, 0.05H), 7.32-7.29 (m, 0.1H), 7.18 (dd, J=9.1, 9.1 Hz,0.95H), 6.93 (dd, J=6.4, 2.7 Hz, 0.95H), 6.71-6.66 (m, 0.95H), 6.33 (brs, 1H), 3.35-3.27 (m, 2H), 3.10-3.06 (m, 2H).

Example 6N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 17step E, usingN′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-chloro-4-fluoroaniline [Aldrich, product #228583] as the startingmaterials. LCMS for C₁₂H₁₅ClFN₆O₄S (M+H)⁺: m/z=393.0. ¹H NMR (400 MHz,DMSO-d₆): δ 11.50 (s, 1H), 8.91 (s, 1H), 7.19 (m, 2H), 6.96 (dd, J=6.7,2.5 Hz, 1H), 6.71 (m, 1H), 6.26 (t, J=6.4 Hz, 1H), 3.32 (m, 2H), 3.13(q, J=5.8 Hz, 2H), 2.89 (s, 3H).

Example 74-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A: 3-[(Diphenylmethylene)amino]propan-1-ol

A solution of 3-amino-1-propanol [Aldrich product #A76400] (2.0 mL, 26mmol) in dichloromethane (79 mL) was treated with benzophenone imine(4.4 mL, 26 mmol) and stirred at 25° C. for 16 h. The reaction mixturewas filtered and the filtrate was concentrated to give the desiredproduct (6.3 g, quantitative) as an oil. This material was used withoutfurther purification. LCMS for C₁₆H₁₈NO (M+H)⁺: m/z=240.2.

Step B:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onetrifluoroacetate

A solution of 3-[(diphenylmethylene)amino]propan-1-ol (80 mg, 0.33 mmol)and triphenylphosphine (93 mg, 0.36 mmol) in tetrahydrofuran (1 mL) at0° C. was treated with diisopropyl azodicarboxylate (75 μL, 0.38 mmol)dropwise. The reaction mixture was stirred at 0° C. for 15 min, treatedwith a solution ofN-{4-[4-(3-chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide(100 mg, 0.25 mmol) in tetrahydrofuran (0.5 mL), and stirred at 25° C.for 16 h. The reaction mixture was treated with trifluoroacetic acid (1mL), stirred at 25° C. for 3 h, and concentrated to a crude residue.This material was purified by preparative LCMS to give the desiredproduct (18 mg, 15%). LCMS for C₁₃H₁₃ClFN₆O₃ (M+H)⁺: m/z=355.1

Step C:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example15, step G, using3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onetrifluoroacetate as the starting material in 34% yield. LCMS forC₁₂H₁₆ClFN₇O₄S (M+H)⁺: m/z=408.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.90 (s,1H), 7.20 (dd, J=9.2, 9.0 Hz, 1H), 6.96 (dd, J=6.4, 2.7 Hz, 1H),6.72-6.69 (m, 1H), 6.55 (t, J=6.0 Hz, 1H), 6.51 (s, 2H), 6.16 (t, J=5.9Hz, 1H), 3.28-3.21 (m, 2H), 2.93-2.87 (m, 2H), 1.76-1.72 (m, 2H).

Example 8N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 4,step E, usingN′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide[made according to Example 4, steps A through D] and3-chloro-4-fluoroaniline [Aldrich, product #228583] as the startingmaterials in 10% yield. LCMS for C₁₃H₁₇ClFN₆O₄S (M+H)⁺: m/z=407.1. ¹HNMR (400 MHz, CD₃OD): δ 7.06 (t, J=8.9 Hz, 1H), 6.98 (m, 1H), 6.80 (m,1H), 3.73 (m, 2H), 3.28 (m, 2H), 2.94 (s, 3H), 1.28 (m, 2H).

Example 94-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A:N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example13, step A, usingN-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride [made according to Example 1, steps A through E] and3-trifluoromethyl-4-fluoroaniline [Aldrich, product #217778] as thestarting materials in quantitative yield. LCMS for C₁₃H₁₄F₄N₅O₃ (M+H)⁺:m/z=364.0. ¹H NMR (400 MHz, CD₃OD): δ 7.15 (m, 2H), 7.08 (m, 1H), 3.60(t, J=5.3 Hz, 2H), 3.46 (t, J=5.3 Hz, 2H), 3.38 (s, 3H).

Step B:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example13, step B, usingN-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in 79% yield. LCMS for C₁₄H₁₂F₄N₅O₄ (M+H)⁺:m/z=390.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.20 (dd, J=6.3, 2.4 Hz, 1H),8.03 (m, 1H), 7.76 (t, J=9.5 Hz, 1H), 6.41 (t, J=5.7 Hz, 1H), 3.49 (t,J=5.5 Hz, 2H), 3.39 (q, J=5.7 Hz, 2H), 3.25 (s, 3H).

Step C:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example13, step C, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 99% yield. LCMS for C₁₃H₁₀F₄N₅O₄ (M+H)⁺:m/z=376.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (m, 1H), 8.05 (m, 1H), 7.76(t, J=9.9 Hz, 1H), 6.34 (t, J=5.7 Hz, 1H), 4.87 (t, J=5.2 Hz, 1H), 3.56(q, J=5.5 Hz, 2H), 3.29 (q, J=5.7 Hz, 2H).

Step D:2-[(4-{4-[4-Fluoro-3-(trifluoromethyl)phenyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

The desired compound was prepared according to the procedure of Example13, step D, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 95% yield. LCMS for C₁₄H₁₂F₄N₅O₆S (M+H)⁺:m/z=454.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.23 (dd, J=6.5, 2.5 Hz, 1H),8.06 (m, 1H), 7.76 (t, J=9.6 Hz, 1H), 6.76 (t, J=5.8 Hz, 1H), 4.37 (t,J=5.4 Hz, 2H), 3.60 (q, J=5.5 Hz, 2H), 3.17 (s, 3H).

Step E:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example13, step E, using2-[(4-{4-[4-fluoro-3-(trifluoromethyl)phenyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate as the starting material in 100% yield. LCMS forC₁₃H₉F₄N₆O₃ (M−N₂+H)⁺: m/z=372.8. ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (dd,J=6.2, 2.4 Hz, 1H), 8.05 (m, 1H), 7.76 (t, J=9.6 Hz, 1H), 6.75 (t, J=5.9Hz, 1H), 3.53 (t, J=5.9 Hz, 2H), 3.45 (q, J=5.6 Hz, 2H).

Step F:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example13, step F, using3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 80% yield. LCMS for C₁₃H₁₁F₄N₆O₃ (M+H)⁺:m/z=375.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.20 (dd, J=6.2, 2.4 Hz, 1H),8.03 (m, 1H), 7.74 (t, J=9.8 Hz, 1H), 7.10 (br s, 0.4H), 6.68 (t, J=5.5Hz, 1H), 3.42 (q, J=5.8 Hz, 2H), 2.95 (t, J=6.5 Hz, 2H).

Step G:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example13, step G, using3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide as the starting material in 55% yield. LCMS forC₁₂H₁₄F₄N₇O₄S (M+H)⁺: m/z=428.0. ¹H NMR (400 MHz, DMSO-d₆): δ 11.60 (s,1H), 9.06 (s, 1H), 7.30 (t, J=10.1 Hz, 1H), 7.14 (dd, J=6.1, 2.7 Hz,1H), 7.03 (m, 1H), 6.71 (t, J=5.3 Hz, 1H), 6.58 (s, 2H), 6.23 (t, J=6.2Hz, 1H), 3.36 (q, J=6.5 Hz, 2H), 3.08 (m, 2H).

Example 10N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 17step E, usingN′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-trifluoromethyl-4-fluoroaniline [Aldrich, product #217778] as thestarting materials. LCMS for C₁₃H₁₅F₄N₆O₄S (M+H)⁺: m/z=427.0. ¹H NMR(400 MHz, DMSO-d₆): δ 11.60 (s, 1H), 9.07 (s, 1H), 7.30 (t, J=10.1 Hz,1H), 7.18 (t, J=6.0 Hz, 1H), 7.13 (dd, J=6.0, 2.7 Hz, 1H), 7.03 (m, 1H),6.27 (t, J=6.3 Hz, 1H), 3.32 (m, 2H), 3.13 (q, J=6.0 Hz, 2H), 2.89 (s,3H).

Example 114-({3-[(Aminosulfonyl)amino]propyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A:4-Amino-N′-hydroxy-N-(3-methoxypropyl)-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example1, step C, using 3-methoxy-1-propanamine as the starting material in 93%yield. LCMS for C₇H₁₄N₅O₃ (M+H)⁺: m/z=216.1.

Step B:N′-Hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example1, step D, using4-amino-N′-hydroxy-N-(3-methoxypropyl)-1,2,5-oxadiazole-3-carboximidamideas the starting material in 72% yield. LCMS for C₇H₁₄N₅O₃ (M+H)⁺:m/z=216.1. ¹H NMR (300 MHz, DMSO-d₆): δ10.4 (s, 1H), 6.21-6.13 (m, 3H),3.37 (t, J=6.1 Hz, 2H), 3.28-3.21 (m, 5H), 1.82-1.74 (m, 2H).

Step C:N-Hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride

The desired compound was prepared according to the procedure of Example1, step E, usingN′-Hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in quantitative yield. LCMS for C₇H₁₂ClN₄O₃(M+H)⁺: m/z=235.1.

Step D:N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example1, step F, usingN-hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride and 4-fluoro-3-(trifluoromethyl)benzeneamine as the startingmaterials in 87% yield. LCMS for C₁₄H₁₆F₄N₅O₃ (M+H)⁺: m/z=378.1. ¹H NMR(400 MHz, DMSO-d₆): δ 11.5 (s, 1H), 9.05 (s, 1H), 7.30 (dd, J=10.0, 9.6Hz, 1H), 7.13-7.11 (m, 1H), 7.05-7.00 (m, 1H), 6.22 (t, J=5.7 Hz, 1H),3.35-3.32 (m, 2H), 3.25-3.19 (m, 5H), 1.79-1.72 (m, 2H).

Step E:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example1, step G, usingN-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in quantitative yield. LCMS for C₁₅H₁₄F₄N₅O₄(M+H)⁺: m/z=404.0.

Step F:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-hydroxypropy)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example3, step D, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 97% yield. LCMS for C₁₄H₁₂F₄N₅O₄ (M+H)⁺:m/z=390.0. ¹H NMR (300 MHz, DMSO-d₆): δ 8.20 (dd, J=6.4, 2.6 Hz, 1H),8.06-8.01 (m, 1H), 7.75 (dd, J=10.0, 9.4 Hz, 1H), 6.53 (t, J=5.7 Hz,1H), 4.59 (t, J=5.0 Hz, 1H), 3.51-3.42 (m, 2H), 3.32-3.26 (m, 2H),1.73-1.68 (m, 2H).

Step G:3-{4-[(3-Azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example3, step E, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in quantitative yield. LCMS for C₁₄H₁₀F₄N₈O₃Na(M+Na)⁺: m/z=437.0.

Step H:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example3, step F, using3-{4-[(3-azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 81% yield. LCMS for C₁₄H₁₃F₄N₆O₃ (M+H)⁺:m/z=389.1. ¹H NMR (300 MHz, DMSO-d₆): δ 8.18 (dd, J=6.4, 2.3 Hz, 1H),8.06-8.01 (m, 1H), 7.72 (dd, J=9.7, 9.4 Hz, 1H), 7.34 (br s, 2H), 6.71(br s, 1H), 2.78-2.73 (m, 2H), 1.85-1.75 (m, 2H).

Step I:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example15, step G, using3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide as the starting material in 60% yield. LCMS forC₁₃H₁₆F₄N₇O₄S (M+H)⁺: m/z=442.0. ¹H NMR (300 MHz, DMSO-d₆): δ 11.6 (s,1H), 9.08 (s, 1H), 7.31 (dd, J=10.0, 9.4 Hz, 1H), 7.13 (dd, J=6.4, 2.9Hz, 1H), 7.05-6.99 (m, 1H), 6.58 (t, J=6.0 Hz, 1H), 6.52 (s, 2H), 6.17(t, J=5.9 Hz, 1H), 3.28-3.21 (m, 2H), 2.94-2.87 (m, 2H), 1.79-1.72 (m,2H).

Example 12N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example16 using3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one hydroiodide as the startingmaterial in 70% yield. LCMS for C₁₄H₁₇F₄N₆O₄S (M+H)⁺: m/z=441.1. ¹H NMR(400 MHz, DMSO-d₆): δ 11.6 (s, 1H), 9.07 (s, 1H), 7.30 (dd, J=10.0, 9.6Hz, 1H), 7.13 (dd, J=6.2, 2.5 Hz, 1H), 7.05-7.02 (m, 2H), 6.19 (t, J=5.8Hz, 1H), 3.27-3.21 (m, 2H), 2.99-2.94 (m, 2H), 2.87 (s, 3H), 1.76-1.72(m, 2H).

Example 134-({2-[(Aminosulfonyl)amino]ethyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

Step A:N′-hydroxy-4-[(2-methoxyethyl)amino]-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride (1.3 g, 5.0 mmol) [made according to Example 1, steps A throughE] was stirred in water (10 mL) and warmed to 60° C. for 5 minutes.3-(trifluoromethyl)aniline [Aldrich, product #A41801] (880 mg, 5.5 mmol)was added in one portion and the reaction stirred for 15 minutes. Whileremaining at 60° C., a solution of sodium bicarbonate (630 mg, 7.5 mmol)in water (10 mL) was added dropwise over 5 minutes. The reaction wasstirred at 60° C. for an additional 50 minutes, and then allowed to coolto room temperature. Ethyl acetate (20 mL) and brine (30 mL) were addedto the flask and the organic layer was collected. The aqueous layer wasextracted with ethyl acetate (2×20 mL) and the combined organics weredried over sodium sulfate. The solvent was removed in vacuo to give thedesired product as an orange solid (1.4 g, 80%). LCMS calculated forC₁₃H₁₅F₃N₅O₃ (M+H)⁺: m/z=346.1. ¹H NMR (400 MHz, CD₃OD): δ 7.36 (t,J=8.2 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 7.09 (s, 1H), 7.02 (d, J=8.2 Hz,1H), 3.60 (t, J=5.2 Hz, 2H), 3.46 (t, J=5.2 Hz, 2H), 3.38 (s, 3H).

Step B:3-{4-[(2-Methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

N′-Hydroxy-4-[(2-methoxyethyl)amino]-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide(1.4 g, 3.80 mmol) and 1,1′-carbonyldiimidazole (1.16 g, 7.16 mmol) weredissolved in ethyl acetate (20 mL). The reaction mixture was heated at70° C. for 40 minutes. Additional 1,1′-carbonyldiimidazole (0.26 g, 1.16mmol) was added. After stirring at 70° C. for another 50 minutes, thereaction was allowed to cool to room temperature. Ethyl acetate (20 mL)was added and the crude reaction was washed with 1 N HCl in water (2×20mL). Brine was added to aid in the separation of the first wash. Theorganic layer was dried over sodium sulfate and concentrated in vacuo.Purification by flash chromatography on silica gel with an eluent ofethyl acetate in hexanes gave the desired product (1.3 g, 90%). LCMScalculated for C₁₄H₁₃F₃N₅O₄ (M+H)⁺: m/z=372.0. ¹H NMR (400 MHz,DMSO-d₆): δ 8.07 (s, 1H), 7.92 (m, 2H), 7.79 (t, J=8.1 Hz, 1H), 6.42 (t,J=6.0 Hz, 1H), 3.47 (t, J=5.8 Hz, 2H), 3.38 (q, J=5.0 Hz, 2H), 3.24 (s,3H).

Step C:3-{4-[(2-Hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

In a round bottom flask under nitrogen atmosphere,3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one(1.3 g, 3.6 mmol) was stirred in dichloromethane (11 mL). Thetemperature was brought to −78° C. and a solution of 1.0 M borontribromide in dichloromethane (7.9 mL, 7.9 mmol) was added dropwise over15 minutes. The reaction was warmed to room temperature over 45 minutesand continued to stir at room temperature for an additional 45 minutes.The reaction was cooled to 0° C. and a saturated solution of sodiumbicarbonate in water (25 mL) was added dropwise over 15 minutes. Afterwarming to room temperature, ethyl acetate (10 mL) and water (10 mL)were added to the flask. The organic layer was collected and the aqueouslayer was extracted with ethyl acetate (2×20 mL). After drying thecombined organic layers over sodium sulfate, the solvent was removed invacuo to give the desired product (1.0 g, 81%). LCMS calculated forC₁₃H₁₁F₃N₅O₄ (M+H)⁺: m/z=358.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (s,1H), 7.93 (t, J=8.2 Hz, 2H), 7.79 (t, J=8.2 Hz, 1H), 6.35 (t, J=5.7 Hz,1H), 4.86 (br s, 1H), 3.55 (t, J=6.0 Hz, 2H), 3.28 (m, 2H).

Step D:2-[(4-{5-Oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

To a solution of3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one(1.0 g, 2.9 mmol) in ethyl acetate (8.5 mL) was added methanesulfonylchloride (0.29 mL, 3.7 mmol) in one portion. The reaction was stirredfor 5 minutes and triethylamine (0.52 mL, 3.7 mmol) was added, also inone portion. After stirring for an additional 10 minutes, the reactionwas quenched with the addition of water (5 mL). The product wasextracted with ethyl acetate (2×5 mL), dried over sodium sulfate andconcentrated in vacuo to give the desired product (1.2 g, 99%). LCMScalculated for C₁₄H₁₃F₃N₅O₆S (M+H)⁺: m/z=436.0. ¹H NMR (400 MHz,DMSO-d₆): δ 8.10 (s, 1H), 7.92 (m, 2H), 7.80 (t, J=8.2 Hz, 1H), 6.77 (t,J=5.9 Hz, 1H), 4.36 (t, J=5.5 Hz, 2H), 3.58 (m, 2H), 3.17 (s, 3H).

Step E:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

2-[(4-{5-Oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate (1.2 g, 2.9 mmol) was dissolved inN,N-dimethylformamide (2.7 mL). After sodium azide (280 mg, 4.3 mmol)was added in one portion, the temperature was brought to 65° C. and thereaction stirred for 6 hours. After cooling back to room temperature,water (10 mL) was added to quench the reaction. The product wasextracted with ethyl acetate (3×10 mL) and the combined organic layerswere dried over sodium sulfate. The solvent was removed in vacuo to givethe desired product (1.05 g, 96%). LCMS calculated for C₁₃H₁₀F₃N₆O₃(M−N₂+H)⁺: m/z=355.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.93(m, 2H), 7.79 (t, J=8.2 Hz, 1H), 6.75 (t, J=5.8 Hz, 1H), 3.52 (t, J=5.7Hz, 2H), 3.44 (q, J=5.5 Hz, 2H).

Step F:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

To a solution of3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one(1.05 g, 2.8 mmol) in methanol (12 mL) was added sodium iodide (2.5 g,17 mmol). After stirring for 10 minutes, a solution ofchlorotrimethylsilane (2.1 mL, 17 mmol) in methanol (1.41 mL) was addeddropwise over 15 minutes. The reaction continued to stir for 40 minutesand then a solution of sodium thiosulfate (2.7 g, 17 mmol) in water(12.5 mL) was added in one portion. A beige solid precipitated uponaddition of the sodium thiosulfate solution and it was collected byvacuum filtration. The solid was rinsed with water (2×10 mL) and wasdried under vacuum overnight to give the desired product. A solid hadalso precipitated from the filtrate and it was collected by vacuumfiltration. After washing with water (3×10 mL) in the funnel, theproduct was dried overnight under vacuum. The solid was slurry washedwith ethyl acetate (3.8 mL) for 1 hour and recollected by filtration.After rinsing with ethyl acetate (2×2 mL) and drying overnight,additional product was obtained. In total, 760 mg of desired product(57%) was obtained as the hydroiodide salt. LCMS calculated forC₁₃H₁₂F₃N₆O₃ (M+H)⁺: m/z=357.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.10 (s,1H), 7.95 (m, 2H), 7.81 (t, J=8.1 Hz, 1H), 7.68 (br s, 2H), 6.74 (t,J=6.7 Hz, 1H), 3.49 (m, 2H), 3.03 (t, J=6.7 Hz, 2H).

Step G:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

To a solution of chlorosulfonyl isocyanate (9.2 μL, 0.11 mmol) indichloromethane (0.24 mL), at 0° C. and under a nitrogen atmosphere, wasadded tert-butyl alcohol (10 μL, 0.11 mmol) in a dropwise fashion. Thesolution was allowed to stir at room temperature for 1 hour to obtain asolution of tert-butyl[chlorosulfonyl]carbamate.

In a separate flask,3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (26 mg, 0.053 mmol) was suspended in dichloromethane (0.5mL). A nitrogen atmosphere was established and the temperature broughtto 0° C. The tert-butyl[chlorosulfonyl]carbamate solution (prepared asabove) was added over 5 minutes to the stirred suspension of the aminesalt. After 10 minutes, triethylamine (37 μL, 0.27 mmol) was addeddropwise. The reaction mixture was stirred at room temperature for 1.5hours. After concentrating in vacuo, the residue was treated withtrifluoroacetic acid (0.5 mL, 6 mmol). This was stirred for 1 hour andthe mixture was again concentrated to dryness in vacuo. The dried solidswere suspended in methanol (0.5 mL) and a 2.0 N NaOH in water (0.53 mL,1.1 mmol) was added in one portion. The reaction was heated to 45° C.and stirred for 30 minutes. After neutralization with acetic acid (60μL, 1.1 mmol), the product was purified by preparative LCMS to give thedesired product (8.5 mg, 39%). LCMS calculated for C₁₂H₁₅F₃N₇O₄S (M+H)⁺:m/z=410.0. ¹H NMR (400 MHz, CD₃OD): δ 7.36 (t, J=7.8 Hz, 1H), 7.23 (d,J=7.8 Hz, 1H), 7.10 (s, 1H), 7.03 (d, J=7.8 Hz, 1H), 3.48 (m, 2H), 3.29(m, 2H).

Example 14N′-Hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example17, step E, usingN′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-trifluoromethylaniline [Aldrich, product #A41801] as the startingmaterials. LCMS for C₁₃H₁₆F₃N₆O₄S (M+H)⁺: m/z=409.1. ¹H NMR (500 MHz,DMSO-d₆): δ 11.63 (s, 1H), 9.08 (s, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.21(m, 2H), 7.10 (s, 1H), 6.99 (d, J=8.1 Hz, 1H), 6.28 (t, J=5.4 Hz, 1H),3.36 (q, J=5.8 Hz, 2H), 3.17 (q, J=5.8 Hz, 2H), 2.91 (s, 3H).

Example 154-({3-[(Aminosulfonyl)amino]propyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

Step A:3-(4-Amino-1,2,5-oxadiazol-3-yl)-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example5, step A, using4-amino-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide[see US Pat. App. Pub. No. 2006/0258719] as the starting material in 97%yield. LCMS for C₁₁H₇F₃N₅O₃ (M+H)⁺: m/z=314.1.

Step B:2,2,2-Trifluoro-N-(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)acetamide

The desired compound was prepared according to the procedure of Example5, step B, using3-(4-amino-1,2,5-oxadiazol-3-yl)-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 90% yield. LCMS for C₁₃H₆F₆N₅O₄ (M+H)⁺:m/z=410.0. ¹H NMR (400 MHz, DMSO-d₆): δ 7.91-7.88 (m, 2H), 7.76-7.69 (m,2H).

Step C:3-{4-[(3-Methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example3, step C, using2,2,2-trifluoro-N-(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)acetamideas the starting material in 49% yield. LCMS for C₁₅H₁₅F₃N₅O₄ (M+H)⁺:m/z=386.1. ¹H NMR (300 MHz, CDCl₃): δ 7.83 (d, J=8.1 Hz, 1H), 7.72-7.67(m, 2H), 7.59 (d, J=7.5 Hz, 1H), 6.08-6.04 (m, 1H), 3.57 (t, J=5.6 Hz,2H), 3.54-3.47 (m, 2H), 3.40 (s, 3H), 2.01-1.93 (m, 2H).

Step D:3-{4-[(3-Hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example3, step D, using3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 69% yield. LCMS for C₁₄H₁₃F₃N₅O₄ (M+H)⁺:m/z=372.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.07 (s, 1H), 7.95-7.90 (m, 2H),7.79 (dd, J=7.9, 7.9 Hz, 1H), 6.55 (t, J=5.6 Hz, 1H), 4.59 (t, J=5.1 Hz,1H), 3.47-3.42 (m, 2H), 3.30-3.25 (m, 2H), 1.72-1.65 (m, 2H).

Step E:3-{4-[(3-Azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example3, step E, using3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 92% yield. LCMS for C₁₄H₁₁F₃N₈O₃Na (M+Na)⁺:m/z=419.0.

Step F:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example3, step F, using3-{4-[(3-azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 92% yield. LCMS for C₁₄H₁₄F₃N₆O₃ (M+H)⁺:m/z=371.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.96-7.92 (m, 2H),7.80 (dd, J=8.0, 7.8 Hz, 1H), 7.53 (br s, 2H), 6.70-6.65 (m, 1H), 4.10(br s, 1H), 3.32-3.31 (m, 2H), 2.81-2.78 (m, 2H), 1.85-1.82 (m, 2H).

Step G:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

A solution of3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (1.5 g, 3.0 mmol) and sulfamide (1.7 g, 18 mmol) in pyridine(60 mL) was heated in a microwave at 130° C. for 10 min. The reactionmixture was concentrated to give the crude intermediateN-{3-[(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]propyl}sulfamide.A solution of the crude intermediate in methanol (90 mL) was treatedwith 2 N NaOH (12 mL, 24 mmol) and stirred at 25° C. for 30 min. Thereaction mixture was treated with 6 M HCl until the solution was acidicand extracted with ethyl acetate (250 mL). The organic layer was washedwith water (100 mL) and brine (100 mL), dried over anhydrous sodiumsulfate, filtered, and concentrated to give a crude residue. Thismaterial was purified by preparative LCMS to give the desired product(1.1 g, 82%) as a gummy solid. LCMS for C₁₃H₁₇F₃N₇O₄S (M+H)⁺: m/z=424.0.¹H NMR (400 MHz, DMSO-d₆): δ 11.6 (s, 1H), 9.12 (s, 1H), 7.37 (dd,J=8.0, 8.0 Hz, 1H), 7.21-7.18 (m, 1H), 7.07 (s, 1H), 6.95 (d, J=10.0 Hz,1H), 6.52 (br s, 3H), 6.17 (t, J=6.0 Hz, 1H), 3.28-3.22 (m, 2H),2.93-2.89 (m, 2H), 1.77-1.73 (m, 2H).

Example 16N′-Hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

A solution of3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (from Example 15, Step F; 25 mg, 50 μmol) in dichloromethane(1 mL) was treated with triethylamine (17 μL, 0.12 mmol) andmethanesulfonyl chloride (6 μL, 70 μmol) and stirred at 25° C. for 2 h.The reaction mixture was concentrated to give the intermediate,N-{3-[(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]propyl}methanesulfonamide,as a crude residue which was used without further purification. Asolution of the crude intermediate in methanol (1 mL) was treated with 2N NaOH (0.25 mL, 0.5 mmol) and stirred at 25° C. for 30 min. Thereaction mixture was treated with acetic acid (50 μL, 0.9 mmol),filtered and purified by preparative LCMS to give the desired product(13 mg, 65%) as a solid. LCMS for C₁₄H₁₈F₃N₆O₄S (M+H)⁺: m/z=423.1. ¹HNMR (400 MHz, DMSO-d₆): δ 11.6 (s, 1H), 9.11 (s, 1H), 7.37 (dd, J=8.0,8.0 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 7.07-7.01 (m, 2H), 6.96 (d, J=8.0Hz, 1H), 6.20 (t, J=5.9 Hz, 1H), 3.27-3.22 (m, 2H), 2.99-2.94 (m, 2H),2.87 (s, 3H), 1.78-1.71 (m, 2H).

Example 17N-(4-Fluoro-3-methylphenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

Step A: tert-Butyl {2-[(methylsulfonyl)amino]ethyl}carbamate

N-(2-Aminoethyl)(tert-butoxy)carboxamide (17.5 mL, 0.11 mol) [Alfa#L19947] was stirred in dichloromethane (320 mL) and triethylamine (33mL, 0.24 mol) was added. A solution of methanesulfonyl chloride (8.5 mL,0.11 mol) in dichloromethane (10 mL) was added. The resulting mixturewas stirred for 1 hour and water (30 mL) was added. The product wasextracted with dichloromethane (3×30 mL), dried over sodium sulfate andconcentrated in vacuo to give the desired product (21 g, 81%). LCMScalculated for C₃H₁₁N₂O₂S (M−Boc+H)⁺: m/z=139.1.

Step B: N-(2-Aminoethyl)methanesulfonamide dihydrochloride

tert-Butyl {2-[(methylsulfonyl)amino]ethyl}carbamate (21 g, 88 mmol) wasstirred in a solution of 4 N hydrogen chloride in 1,4-dioxane (97 mL,388 mmol) for 30 minutes. Trituration with ethyl acetate and hexanesfollowed by diethyl ether and hexanes gave the desired compound as a gum(19 g, 100%). LCMS calculated for C₃H₁₁N₂O₂S (M+H)⁺: m/z=139.0.

Step C:4-Amino-N′-hydroxy-N-{2-[(methylsulfonyl)amino]ethyl}-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (9.7 g, 60mmol) was stirred in ethanol (460 mL) andN-(2-aminoethyl)methanesulfonamide dihydrochloride (19 g, 109 mmol) wasadded slowly in portions and the temperature rose to 25° C. Aftercooling back to 0° C., triethylamine (53 mL, 380 mmol) was addeddropwise over 15 minutes and the reaction was stirred for an additional15 minutes. The solution was washed with water (300 mL) and brine (300mL). The organic layer was dried over sodium sulfate and concentrated invacuo to give the desired product (16 g, 100%). LCMS calculated forC₆H₁₃N₆O₄S (M+H)⁺: m/z=265.1. ¹H NMR (400 MHz, DMSO-d₆): δ 10.16 (s,1H), 9.07 (m, 1H), 7.18 (m, 1H), 6.37 (s, 2H), 3.36 (m, 2H), 3.15 (m,2H), 2.87 (s, 3H).

Step D:N′-Hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N′-hydroxy-N-{2-[(methylsulfonyl)amino]ethyl}-1,2,5-oxadiazole-3-carboximidamide(0.47 g, 1.8 mmol) was stirred in 1,2-ethanediol (38 mL). Potassiumhydroxide (600 g, 11 mmol) was added in one portion. The reaction washeated at 130° C. for 4 hours and allowed to cool to room temperature. 1N HCl solution (60 mL) was added and the product was extracted withethyl acetate (4×40 mL). The combined organics were dried over sodiumsulfate and concentrated in vacuo to give the desired product (0.45 g,96%). LCMS calculated for C₆H₁₂N₆O₄S (M+H)⁺: m/z=265.1. ¹H NMR (400 MHz,DMSO-d₆): δ 10.49 (s, 1H), 7.18 (m, 1H), 6.20 (m, 3H), 3.36 (m, 2H),3.15 (m, 2H), 2.87 (s, 3H).

Step E:N-(4-Fluoro-3-methylphenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

N′-Hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide(35 mg, 0.13 mmol) was stirred in 1,4-dioxane (2 mL) and 6 N hydrogenchloride solution (4 mL) was added. The solution was cooled to 0° C. anda solution of sodium nitrite (11 mg, 0.16 mmol) in water (3 mL) wasslowly added. The mixture was stirred for 1 hour at 0° C. andevaporated. Dry 1,4-dioxane (2 mL) was added and the mixture evaporatedtwo more times. A solution of 4-fluoro-3-methylaniline [Aldrich, product#559415] (25 mg, 0.20 mmol) in ethanol (2 mL) was added and the mixturewas stirred for 1 hour. Purification by preparative LCMS (pH 2) gave thedesired compound (17 mg, 27%). LCMS calculated for C₁₃H₁₅FN₆O₄S (M+H)⁺:m/z=373.1. ¹H NMR (400 MHz, DMSO-d₆): δ 11.25 (s, 1H), 8.61 (s, 1H),7.18 (m, 1H), 6.91 (m, 1H), 6.72 (m, 1H), 6.58 (m, 1H), 6.24 (s, 1H),3.32 (m, 2H), 3.11 (m, 2H), 2.89 (s, 3H), 2.05 (s, 3H).

Example 184-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-cyano-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A:N-(3-Cyano-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example13, step A, usingN-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride [made according to Example 1, steps A through E] and5-amino-2-fluorobenzonitrile [Aldrich, product #639877] as the startingmaterials in 100% yield. LCMS for C₁₃H₁₄FN₆O₃ (M+H)⁺: m/z=321.0.

Step B:2-Fluoro-5-[3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrile

The desired compound was prepared according to the procedure of Example13, step B, usingN-(3-cyano-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in 91% yield. LCMS for C₁₄H₁₂FN₆O₄ (M+H)⁺:m/z=347.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.25 (dd, J=5.7, 2.6 Hz, 1H),8.06 (m, 1H), 7.77 (t, J=9.2 Hz, 1H), 6.41 (t, J=5.7 Hz, 1H), 3.48 (m,2H), 3.40 (q, J=5.4 Hz, 2H), 3.25 (s, 3H).

Step C:2-Fluoro-5-[3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrile

The desired compound was prepared according to the procedure of Example13, step C, using2-fluoro-5-[3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrileas the starting material in quantitative yield. LCMS for C₁₃H₁₀FN₆O₄(M+H)⁺: m/z=333.0.

Step D:2-({4-[4-(3-Cyano-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate

The desired compound was prepared according to the procedure of Example13, step D, using2-fluoro-5-[3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrileas the starting material in 88% yield. LCMS for C₁₄H₁₂FN₆O₆S (M+H)⁺:m/z=411.0.

Step E:5-[3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrile

The desired compound was prepared according to the procedure of Example13, step E, using2-({4-[4-(3-cyano-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate as the starting material in 95% yield.

Step F:5-[3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrilehydroiodide

The desired compound was prepared according to the procedure of Example13, step F, using5-[3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrileas the starting material in 57% yield. LCMS for C₁₃H₁₁FN₇O₃ (M+H)⁺:m/z=332.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.29 (dd, J=5.8, 2.7 Hz, 1H),8.09 (m, 1H), 7.83 (br s, 3H), 7.79 (t, J=9.0 Hz, 1H), 6.77 (t, J=5.9Hz, 1H), 3.50 (q, J=6.4 Hz, 2H), 3.04 (m, 2H).

Step G:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-cyano-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

In a microwave vial,5-[3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrilehydroiodide (20.0 mg, 0.044 mmol) and sulfamide (25 mg, 0.26 mmol) weresuspended in pyridine (0.5 mL). The reaction was heated to 120° C. for10 minutes in a microwave reactor. The solvent was removed and theresidue dissolved in methanol (0.17 mL). A solution of 2.0 N NaOH inwater (0.22 mL, 0.44 mmol) was added in one portion. The reaction wasstirred at room temperature overnight. After neutralization with aceticacid (50 μL), the product was purified using preparative LCMS to givethe title compound (4.9 mg, 29%). LCMS for C₁₂H₁₄FN₈O₄S (M+H)⁺:m/z=385.0. ¹H NMR (400 MHz, DMSO-d₆): δ 11.65 (s, 1H), 9.08 (s, 1H),7.34 (t, J=9.1 Hz, 1H), 7.22 (dd, J=5.4, 2.8 Hz, 1H), 7.13 (m, 1H), 6.70(t, J=5.9 Hz, 1H), 6.59 (s, 2H), 6.20 (t, J=6.1 Hz, 1H), 3.34 (m, 2H),3.09 (m, 2H).

Example 19N-(3-Cyano-4-fluorophenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example17, step E, usingN′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-cyano-4-fluoroaniline [Aldrich, product #639877] as the startingmaterials. LCMS for C₁₃H₁₄FN₇NaO₄S (M+Na)⁺: m/z=406.0. ¹H NMR (400 MHz,DMSO-d₆): δ 11.65 (s, 1H), 9.08 (s, 1H), 7.35 (m, 1H), 7.18 (m, 3H),6.56 (m, 1H), 6.23 (m, 1H), 6.24 (s, 2H), 3.32 (m, 2H), 3.14 (m, 2H),2.89 (s, 3H).

Example 204-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-bromo-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A: tert-Butyl[(4-bromo-2-furyl)methyl]carbamate

4-Bromo-2-furaldehyde [Aldrich, product #666599] (10.0 g, 57.1 mmol) wasdissolved in ethanol (50 mL) and water (50 mL). N-Hydroxyaminehydrochloride (7.15 g, 103 mmol) and sodium acetate (8.44 g, 103 mmol)were added sequentially and the reaction mixture was brought to refluxat 100° C. for 1 hour. The solution was partially concentrated and theprecipitate was collected and washed with cold water (2×10 mL). Thefiltrate was extracted with ethyl acetate (3×25 mL) and the combinedorganic layers were washed with brine (50 mL). After drying over sodiumsulfate, the solution was concentrated in vacuo. The residue wascombined with the precipitate and dissolved in acetic acid (70 mL).After placing in an ice-bath, zinc (14.7 g, 225 mmol) was addedportion-wise over 25 minutes. The reaction warmed to room temperatureover 1.5 hours and was filtered through Celite. The solvent was removedin vacuo.

The residue was stirred in tetrahydrofuran (72 mL). A solution of 2.0 NNaOH in water (179 mL, 358 mmol) was added dropwise over 45 minutes.After 5 minutes, di-tert-butyldicarbonate (16.9 g, 77.4 mmol) was addeddropwise. The reaction was stirred for 2 hours and the tetrahydrofuranwas removed in vacuo. Ethyl acetate (100 mL) was added and thesuspension was filtered. The organic layer was collected and the productextracted with ethyl acetate (2×50 mL). The combined organic layers werewashed with brine (100 mL) and water (100 mL), dried over sodium sulfateand concentrated in vacuo to give the desired product (15.3 g, 79%).LCMS calculated for C₁₀H₁₄BrNNaO₃ (M+Na)⁺: m/z=298.0. ¹H NMR (400 MHz,DMSO-d₆): δ 7.79 (s, 1H), 7.37 (t, J=5.8 Hz, 1H), 6.33 (s, 1H), 4.06 (d,J=6.1 Hz, 2H), 1.36 (s, 9H).

Step B: 1-(4-Bromo-2-furyl)methanamine trifluoroacetate

Under a nitrogen atmosphere, a solution oftert-butyl[(4-bromo-2-furyl)methyl]carbamate (15.3 g, 55.4 mmol) indichloromethane (86 mL) at 0° C. was treated with trifluoroacetic acid(43 mL) over 15 minutes. The reaction mixture warmed to room temperatureover 30 minutes. The solvent was removed in vacuo and chased withtoluene (3×50 mL). The product was lyophilized for 18 hours to give thedesired product as a brown solid (13.0 g, 81%). LCMS calculated forC₅H₄BrO (M+H₂)⁺: m/z=158.9, 160.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (brs, 3H), 8.01 (s, 1H), 6.70 (s, 1H), 4.08 (s, 1H).

Step C:N-[(4-Bromo-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-(2-methoxyethylamino)-1,2,5-oxadiazole-3-carbimidoylchloride [prepared according to the procedure of Example 1, steps Athrough E] (4.5 g, 20.3 mmol) was stirred in ethanol (20 mL) at roomtemperature. To this, a solution of 1-(4-bromo-2-furyl)methanaminetrifluoroacetate (6.5 g, 22.4 mmol) in ethanol (24 mL) was added and themixture was stirred for 15 minutes. Triethylamine (6.3 mL, 44.8 mmol)was added dropwise over 10 minutes and the reaction was stirred for anadditional 15 minutes. The solvent was removed in vacuo and after addingwater (50 mL), the product was extracted with ethyl acetate (3×50 mL).The combined organic layers were washed with brine (50 mL), dried oversodium sulfate and concentrated to give the desired product (7.5 g,100%). LCMS calculated for C₁₁H₁₅BrN₅O₄ (M+H)⁺: m/z=359.9, 361.9.

Step D:4-[(4-Bromo-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

N-[(4-Bromo-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(7.3 g, 20.4 mmol) and 1,1′-carbonyldiimidazole (5.0 g, 30.5 mmol) weredissolved in ethyl acetate (72 mL). The reaction mixture was heated at65° C. for 15 minutes. Ethyl acetate (70 mL) was added and the crudereaction was washed with 0.1 N hydrogen chloride in water (2×70 mL). Theorganic layer was dried over sodium sulfate and concentrated in vacuo.Purification by flash chromatography on silica gel with an eluent ofethyl acetate in hexanes gave the desired product (4.1 g, 90%). LCMScalculated for C₁₂H₁₃BrN₅O₅ (M+H)⁺: m/z=386.0, 388.0. ¹H NMR (400 MHz,CD₃OD): δ 7.88 (s, 1H), 6.67 (s, 1H), 6.39 (t, J=5.7 Hz, 1H), 5.07 (s,2H), 3.50 (m, 2H), 3.41 (q, J=5.7 Hz, 2H), 3.25 (s, 3H).

Step E:4-[(4-Bromo-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

In a round bottom flask under nitrogen atmosphere,4-[(4-bromo-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(8.2 g, 21 mmol) was stirred in dichloromethane (68 mL). The temperaturewas brought to −78° C. and a solution of 1.0 M boron tribromide indichloromethane (43 mL, 43 mmol) was added dropwise over 45 minutes. Thereaction stirred at −78° C. for 45 minutes and continued to stir at 0°C. for an additional 30 minutes. While remaining at 0° C., a saturatedsolution of sodium bicarbonate in water (120 mL) was added dropwise over25 minutes. After warming to room temperature, the organic layer wascollected and the aqueous layer was extracted with ethyl acetate (2×50mL). The combined organic layers were washed with brine (100 mL), driedover sodium sulfate and concentrated in vacuo to give the desiredproduct (7.7 g, 97%) along with a small amount of3-{4-[(2-bromoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one.LCMS calculated for C₁₁H₁₃BrN₅O₅ (M+H)⁺: m/z=371.7, 374.0. ¹H NMR (400MHz, DMSO-d₆): δ 7.89 (s, 1H), 6.68 (s, 1H), 6.31 (t, J=5.8 Hz, 1H),5.08 (s, 2H), 4.85 (br s, 1H), 3.56 (m, 2H), 3.30 (q, J=5.6 Hz, 2H).

Step F:2-[(4-{4-[(4-Bromo-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

To a solution of4-[(4-bromo-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(7.7 g, 21 mmol, containing also some of the correspondingbromo-compound) in ethyl acetate (100 mL) was added methanesulfonylchloride (0.96 mL, 12 mmol) in one portion. The reaction was stirred for5 minutes and triethylamine (1.6 mL, 11 mmol) was added, also in oneportion. After stirring for 30 minutes, additional methanesulfonylchloride (0.4 mL, 5 mmol) was added, followed 5 minutes later bytriethylamine (0.58 mL, 4.2 mmol). After 15 minutes, the reaction wasquenched with the addition of water (100 mL). The product was extractedwith ethyl acetate (3×50 mL) and the combined organic layers washed withbrine (100 mL). After drying over sodium sulfate, the solvent wasremoved in vacuo to give the desired product (9.3 g, 100%). LCMScalculated for C₁₂H₁₃BrN₅O₇S (M+H)⁺: m/z=449.8, 451.8. ¹H NMR (300 MHz,DMSO-d₆): δ 7.88 (s, 1H), 6.73 (t, J=6.2 Hz, 1H), 6.68 (s, 1H), 5.08 (s,2H), 4.37 (m, 2H), 3.59 (q, J=5.8 Hz, 2H), 3.16 (s, 3H).

Step G:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one

2-[(4-{4-[(4-Bromo-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate (9.1 g, 20 mmol, containing also some of thecorresponding bromo-compound) was dissolved in dimethylformamide (90mL). Sodium azide (1.97 g, 30.3 mmol) was added in one portion and after5 minutes, the temperature was brought to 65° C. The reaction stirredfor 2 hours and was allowed to cool back to room temperature. Water (200mL) was added to quench the reaction. The product was extracted withethyl acetate (3×100 mL) and the combined organic layers were washedwith brine (2×150 mL) and water (150 mL). After drying over sodiumsulfate, the solvent was removed in vacuo to give the desired product(7.7 g, 96%). LCMS calculated for C₁₁H₉BrN₈NaO₄ (M+Na)⁺: m/z=418.7,421.0. ¹H NMR (400 MHz, DMSO-d₆): δ 7.88 (s, 1H), 6.71 (t, J=5.7 Hz,1H), 6.68 (s, 1H), 5.08 (s, 2H), 3.54 (t, J=5.7 Hz, 2H), 3.47 (q, J=5.7Hz, 2H).

Step H:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

To a solution of3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one(7.7 g, 19 mmol) in methanol (80 mL) was added sodium iodide (17.4 g,116 mmol). After stirring for 10 minutes, a solution ofchlorotrimethylsilane (14.8 mL, 116 mmol) was added dropwise over 5minutes. The reaction continued to stir for 1 hour, at which time it wasslowly added to a solution of sodium thiosulfate (23.0 g, 145 mmol) inwater (800 mL) at 0° C., resulting in a precipitate. The flask wasrinsed with methanol (10 mL) and the precipitate was collected throughvacuum filtration. The solid was rinsed with cold water (2×25 mL) andwas dried under vacuum to give the desired product (5.8 g, 60%) as thehydroiodide salt. LCMS calculated for C₁₁H₁₂BrN₆O₄ (M+H)⁺: m/z=370.9,372.8. ¹H NMR (400 MHz, DMSO-d₆): δ 7.86 (s, 1H), 7.36 (br s, 3H), 6.68(t, J=5.8 Hz, 1H), 6.65 (s, 1H), 5.07 (s, 2H), 3.45 (q, J=5.8 Hz, 2H),2.98 (t, J=5.8 Hz, 2H).

Step I:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-bromo-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

In a microwave vial,3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (30 mg, 0.060 mmol) and sulfamide (29 mg, 0.30 mmol) weresuspended in pyridine (1 mL). The reaction mixture was flushed withnitrogen and heated at 130° C. for 3 minutes in a microwave reactor. Thesolvent was removed and the crude intermediate was suspended in methanol(1 mL). A 2.0 N solution of NaOH in water (0.30 mL, 0.60 mmol) was addedin one portion and the reaction was heated to 45° C. for 30 minutes.After neutralization with acetic acid (68 μL, 1.2 mmol), the product waspurified by preparative LCMS to give the desired product (10.4 mg, 41%).LCMS calculated for C₁₀H₁₅BrN₇O₅S (M+H)⁺: m/z=423.9, 426.0. ¹H NMR (400MHz, DMSO-d₆): δ 10.87 (s, 1H), 7.75 (s, 1H), 6.83 (t, J=7.3 Hz, 1H),6.68 (t, J=6.0 Hz, 1H), 6.56 (s, 2H), 6.30 (t, J=6.0 Hz, 1H), 6.23 (s,1H), 4.56 (d, J=7.0 Hz, 2H), 3.32 (q, J=6.3 Hz, 2H), 3.07 (q, J=6.3 Hz,2H).

Example 214-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-chloro-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A: 4-Chloro-2-furaldehyde

To a stirred suspension of aluminum trichloride (29.8 g, 0.223 mol) indichloromethane (200 mL) under nitrogen atmosphere was added2-furancarboxaldehyde (8.44 mL, 0.102 mol) over 15 minutes. Afterstirring for 30 minutes, chlorine was bubbled into the suspension usinga pipette over a time period of 50 minutes. The flask was sealed andleft to stir at room temperature for 90 hours. The reaction mixture wasslowly added to a mixture of ice (500 mL) in a solution of 1.0 Nhydrogen chloride in water (300 mL). The mixture was left to warm toroom temperature over the next hour. The layers were separated and theorganic layer collected. Additional product was extracted withdichloromethane (2×200 mL). The combined organic layers were washed withwater (250 mL) and dried over sodium sulfate. The solvent was removed invacuo to give a crude mixture containing the desired product (14.0 g,100%, 60% purity). ¹H NMR (400 MHz, DMSO-d₆): δ 9.56 (s, 1H), 8.36 (s,1H), 7.71 (s, 1H).

Step B: tert-Butyl[(4-chloro-2-furyl)methyl]carbamate

4-Chloro-2-furaldehyde (14.0 g, 60% purity, 64 mmol) was dissolved inethanol (50 mL) and water (50 mL). N-Hydroxyamine hydrochloride (12.6 g,182 mmol) and sodium acetate (14.9 g, 182 mmol) were added sequentiallyand the reaction mixture was brought to reflux at 100° C. for 1 hour.The solution was partially concentrated then water (25 mL) and ethylacetate (50 mL) were added. The organic layer was collected and theaqueous was extracted with ethyl acetate (2×25 mL). The combined organiclayers were washed with brine (50 mL) and water (50 mL). After dryingover sodium sulfate, the solution was concentrated in vacuo. Theintermediate was suspended in acetic acid (115 mL). The solution wascooled in an ice-bath and zinc (33.1 g, 506 mmol) was added portion-wiseover 20 minutes. The reaction warmed to room temperature over 2 hoursand was filtered through Celite. The solvent was removed in vacuo.

The residue was stirred in tetrahydrofuran (100 mL). A solution of 2.0 MNaOH in water (152 mL, 304 mmol) was added dropwise over 30 minutes. Thereaction mixture was placed in an ice-bath and after 5 minutes,di-tert-butyldicarbonate (24.3 g, 111 mmol) was added dropwise over 15minutes. The reaction was allowed to warm to room temperature over thenext 2 hours and the tetrahydrofuran was then removed in vacuo. Ethylacetate (100 mL) was added and the suspension was filtered. The organiclayer was collected and the aqueous layer extracted with ethyl acetate(2×100 mL). The combined organic layers were washed with a 1:1 mixtureof water/brine (100 mL), dried over sodium sulfate and concentrated invacuo. Purification by flash chromatography on silica gel with an eluentof ethyl acetate in hexanes gave the desired product (3.05 g, 22%). LCMScalculated for C₁₀H₁₄ClNNaO₃ (M+Na)⁺: m/z=253.9. ¹H NMR (400 MHz,DMSO-d₆): δ 7.81 (s, 1H), 7.37 (t, J=5.3 Hz, 1H), 6.32 (s, 1H), 4.05 (d,J=6.0 Hz, 2H), 1.36 (s, 9H).

Step C: 1-(4-Chloro-2-furyl)methanamine trifluoroacetate

The desired compound was prepared according to the procedure of Example20, step B, using tert-butyl[(4-chloro-2-furyl)methyl]carbamate as thestarting material in quantitative yield. LCMS calculated for C₅H₄ClN₅O₄(M+H₂)⁺: m/z=115.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.29 (br s, 3H), 8.04(s, 1H), 6.69 (s, 1H), 4.07 (s, 2H).

Step D:N-[(4-Chloro-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example20, step C, usingN-hydroxy-4-(2-methoxyethylamino)-1,2,5-oxadiazole-3-carbimidoylchloride and 1-(4-chloro-2-furyl)methanamine trifluoroacetate as thestarting material in quantitative yield. LCMS calculated forC₁₁H₁₅ClN₅O₄ (M+H)⁺: m/z=316.0.

Step E:4-[(4-Chloro-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example20, step D, usingN-[(4-chloro-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in 51% yield. LCMS calculated for C₁₂H₁₃ClN₅O₅(M+H)⁺: m/z=342.0.

Step F:4-[(4-Chloro-2-furyl)methyl]-3-[4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example20, step E, using4-[(4-chloro-2-furyl)methyl]-3-[4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in quantitative yield. LCMS calculated forC₁₁H₁₀ClN₅NaO₅ (M+Na)⁺: m/z=349.9.

Step G:2-[(4-{4-[(4-Chloro-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

The desired compound was prepared according to the procedure of Example20, step F, using4-[(4-chloro-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 69% yield. LCMS calculated for C₁₂H₁₃ClN₅O₇S(M+H)+: m/z=405.8.

Step H:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example20, step G, using2-[(4-{4-[(4-chloro-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate as the starting material in quantitative yield. LCMScalculated for C₁₁H₉ClN₈NaO₄ (M+Na)⁺: m/z=374.9.

Step I:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example20, step H, using3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 57% yield. LCMS calculated for C₁₁H₁₂ClN₆O₄(M+H)⁺: m/z=326.9.

Step J:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-chloro-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example20, step I, using3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide as the starting material in 53% yield. LCMS calculated forC₁₀H₁₅ClN₇O₅S (M+H)⁺: m/z=379.9. ¹H NMR (400 MHz, DMSO-d₆): δ 10.88 (s,1H), 7.77 (s, 1H), 6.83 (t, J=6.8 Hz, 1H), 6.68 (t, J=5.9 Hz, 1H), 6.56(s, 2H), 6.30 (t, J=5.9 Hz, 1H), 6.22 (s, 1H), 4.55 (d, 2H), 3.32 (q,J=6.3 Hz, 2H), 3.06 (q, J=6.3 Hz, 2H).

Example 22 Alternate Preparation of the Intermediate3-(4-(2-aminoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide

Step A:4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (can beprepared according to Example 1, steps A-B, 200.0 g, 1.23 mol) was mixedwith ethyl acetate (1.2 L). At 0-5° C. 2-methoxyethylamine [Aldrich,product #143693] (119.0 mL, 1.35 mol) was added in one portion whilestirring. The reaction temperature rose to 41° C. The reaction wascooled to 0-5° C. Triethylamine (258 mL, 1.84 mol) was added. Afterstirring 5 min, LCMS indicated reaction completion. The reactionsolution was washed with water (500 mL) and brine (500 mL), dried oversodium sulfate, and concentrated to give the desired product (294 g,119%) as a crude dark oil. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z=202.3. ¹H NMR(400 MHz, DMSO-d₆): δ 10.65 (s, 1H), 6.27 (s, 2H), 6.10 (t, J=6.5 Hz,1H), 3.50 (m, 2H), 3.35 (d, J=5.8 Hz, 2H), 3.08 (s, 3H).

Step B:N′-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide(248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210g, 3.7 mol) was added. The reaction was refluxed at 100° C. overnight(15 hours). TLC with 50% ethyl acetate (containing 1% ammoniumhydroxide) in hexane indicated reaction completed (product Rf=0.6,starting material Rf=0.5). LCMS also indicated reaction completion. Thereaction was cooled to room temperature and extracted with ethyl acetate(3×1 L). The combined ethyl acetate solution was dried over sodiumsulfate and concentrated to give the desired product (201 g, 81%) as acrude off-white solid. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z=202.3 ¹H NMR (400MHz, DMSO-d₆): δ 10.54 (s, 1H), 6.22 (s, 2H), 6.15 (t, J=5.8 Hz, 1H),3.45 (t, J=5.3 Hz, 2H), 3.35 (m, 2H), 3.22 (s, 3H).

Step C:N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride

At room temperatureN′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueoussolution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) wasadded followed by water (250 mL) and ethyl acetate (250 mL). At 3-5° C.a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at3-8° C. for 2 hours and then room temperature over the weekend. LCMSindicated reaction completed. The reaction solution was extracted withethyl acetate (2×200 mL). The combined ethyl acetate solution was driedover sodium sulfate and concentrated to give the desired product (49.9g, 126%) as a crude white solid. LCMS for C₆H₁₀ClN₄O₃ (M+H)⁺: m/z=221.0.¹H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1H), 5.85 (t, J=5.6 Hz, 1H), 3.50(t, J=5.6 Hz, 2H), 3.37 (dd, J=10.8, 5.6 Hz, 2H), 3.25 (s, 3H).

Step D:N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixturewas heated to 60° C. 3-Bromo-4-fluoroaniline [Oakwood products, product#013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warmsodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was addedover 15 min. The reaction was stirred at 60° C. for 20 min. LCMSindicated reaction completion. The reaction solution was cooled to roomtemperature and extracted with ethyl acetate (2×300 mL). The combinedethyl acetate solution was dried over sodium sulfate and concentrated togive the desired product (76.7 g, 98%) as a crude brown solid. LCMS forC₁₂H₁₄BrFN₅O₃ (M+H)⁺: m/z=374.0, 376.0. ¹H NMR (400 MHz, DMSO-d₆): δ11.55 (s, 1H), 8.85 (s, 1H), 7.16 (t, J=8.8 Hz, 1H), 7.08 (dd, J=6.1,2.7 Hz, 1H), 6.75 (m, 1H), 6.14 (t, J=5.8 Hz, 1H), 3.48 (t, J=5.2 Hz,2H), 3.35 (dd, J=10.8, 5.6 Hz, 2H), 3.22 (s, 3H).

Step E:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A mixture ofN-(3-bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(76.5 g, 0.204 mol), 1,1′-carbonyldiimidazole (49.7 g, 0.307 mol), andethyl acetate (720 mL) was heated to 60° C. and stirred for 20 min. LCMSindicated reaction completed. The reaction was cooled to roomtemperature, washed with 1 N HCl(2×750 mL), dried over sodium sulfate,and concentrated to give the desired product (80.4 g, 98%) as a crudebrown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z=400.0, 402.0. ¹H NMR(400 MHz, DMSO-d₆): δ 7.94 (t, J=8.2 Hz, 1H), 7.72 (dd, J=9.1, 2.3 Hz,1H), 7.42 (m, 1H), 6.42 (t, J=5.7 Hz, 1H), 3.46 (t, J=5.4 Hz, 2H), 3.36(t, J=5.8 Hz, 2H), 3.26 (s, 3H).

Step F:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At −67°C. boron tribromide (37 mL, 0.392 mol) was added over 15 min. Thereaction was warmed up to −10° C. in 30 min. LCMS indicated reactioncompleted. The reaction was stirred at room temperature for 1 hour. At0-5° C. the reaction was slowly quenched with saturated sodiumbicarbonate solution (1.5 L) over 30 min. The reaction temperature roseto 25° C. The reaction was extracted with ethyl acetate (2×500 mL, firstextraction organic layer is on the bottom and second extraction organiclager is on the top). The combined organic layers were dried over sodiumsulfate and concentrated to give the desired product (75 g, 99%) as acrude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z=386.0, 388.0. ¹HNMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.70 (m, 1H),7.68 (t, J=8.7 Hz, 1H), 6.33 (t, J=5.6 Hz, 1H), 4.85 (t, J=5.0 Hz, 1H),3.56 (dd, J=10.6, 5.6 Hz, 2H), 3.29 (dd, J=11.5, 5.9 Hz, 2H).

Step G:2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate

4-(3-bromo-4-fluorophenyl)-3-(4-(2-hydroxyethylamino)-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one(72.2 g, 0.188 mol) was mixed with ethyl acetate (600 mL).Methanesulfonyl chloride (19.2 mL, 0.248 mol) was added followed bytriethylamine (34.9 mL, 0.250 mol). The reaction was stirred at roomtemperature for 5 min. When LCMS indicated completion of reaction(M+H=442), 500 mL of water was added into reaction. The reaction wasextracted with ethyl acetate (2×500 mL). The combined ethyl acetatesolution was washed with brine (500 mL), dried over sodium sulfate andconcentrated to give 85.1 g crude brown solid. ¹H NMR verified thestructure. Crude yield was 97%. LCMS for C₁₃H₁₁BrFN₅O₆SNa (M+Na)⁺:m/z=485.9, 487.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz,1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75 (t, J=5.9 Hz, 1H), 4.36(t, J=5.3 Hz, 2H), 3.58 (dd, J=11.2, 5.6 Hz, 2H), 3.18 (s, 3H).

Step H:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylmethanesulfonate (50.0 g, 0.108 mol) was dissolved in N,N-dimethylformamide (83 mL). Sodium azide (10.5 g, 0.162 mol) was added.The reaction was stirred at 65° C. for 5-6 hours. LCMS indicatedreaction completed (M+Na=435). The reaction was quenched with water (250mL) and extracted with ethyl acetate (2×250 mL). The combined ethylacetate solution was washed with water (250 mL, layer separation wasslow, 100 mL of brine was added to improve the separation), dried oversodium sulfate, and concentrated to give 49.7 g crude brown solid. Crudeyield is 112%. LCMS for C₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z=433.0, 435.0. ¹H NMR(400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58(t, J=8.7 Hz, 1H), 6.75 (t, J=5.7 Hz, 1H), 3.54 (t, J=5.3 Hz, 2H), 3.45(dd, J=11.1, 5.2 Hz, 2H).

Step I:3-(4-(2-aminoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide

3-(4-(2-azidoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(80.0 g, 0.194 mol) was mixed with methanol (800 mL). Sodium iodide(175.0 g, 1.17 mol) was added. The reaction was stirred at roomtemperature for 10 min. Chlorotrimethylsilane (148 mL, 1.17 mol) wasdissolved in methanol (100 mL) and added to the reaction over 30 min.The reaction temperature rose 42° C. The reaction was stirred at roomtemperature for 30 min LCMS indicated reaction completed (M+H=386). Thereaction was quenched with sodium thiosulfate (190.0 g, 1.20 mol) inwater (900 mL). A large amount of solid precipitated. The product wascollected by filtration (filtration speed was slow), rinsed with water(200 mL), and dried on vacuum overnight. The filter cake was slurried inethyl acetate (500 mL) for 30 min. The product was filtered (filtrationspeed is slow) and dried under vacuum over weekend to give 95 g of anoff-white solid. LCMS for C₁₂H₁₁BrFN₆O₃ (M+H)⁺: m/z=384.9, 386.9. ¹H NMR(400 MHz, DMSO-d₆): δ 8.12 (m, 4H), 7.76 (m, 1H), 7.58 (t, J=8.7 Hz,1H), 6.78 (t, J=6.1 Hz, 1H), 3.51 (dd, J=11.8, 6.1 Hz, 2H), 3.02 (m,2H).

Example 23 Alternate preparation of4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step A:4-(3-bromo-4-fluorophenyl)-3-(4-(2-hydroxyethylamino)-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one

To a solution of4-(3-bromo-4-fluorophenyl)-3-(4-(2-methoxyethylamino)-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one(can be prepared according to Example 1, steps A-G; 1232 g, 3.08 mol) indichloromethane (12 L) stirring in a 22 L flask at 0° C. was added borontribromide (354 mL, 3.67 mL) dropwise at a rate so that the temperaturedid not exceed 10° C. After stirring on ice for 1 h, a solution ofsaturated aqueous sodium bicarbonate (2 L) was carefully added at a rateso that the temperature did not exceed 20° C. (addition time 10 min).The resulting mixture was transferred to a 50 L separatory funnel,diluted with water (10 L), and the pH of the aqueous layer adjusted from1 to 8 using solid sodium bicarbonate. The layers were separated, andthe organic layer was washed with water (10 L), and the solvents removedin vacuo to afford a tan solid (24 mol processed in multiple runs, 9.54kg, quant. yield). The material was slurried in 4 volumes of 7:1heptane:ethyl acetate (4×22 L flasks), filtered, and dried to furnishthe title compound as a tan solid (8679 g, 94%). The product was amixture of the hydroxy- and the corresponding bromo-species.

Step B:2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylmethanesulfonate

To a solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(1.5 kg, 3.9 mol, containing also some of the correspondingbromo-compound) in ethyl acetate (12 L) was added methanesulfonylchloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature.Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, duringwhich time the reaction temperature increased to 35° C. After 2 h, thereaction mixture was washed with water (5 L), brine (1 L), dried oversodium sulfate, combined with 3 more reactions of the same size, and thesolvents removed in vacuo to afford the desired product (7600 g,quantitative yield, containing also some of the correspondingbromo-compound, Caution: irritating dust!) as a tan solid. LCMS forC₁₃H₁₁BrFN₅O₆SNa (M+Na)⁺: m/z=485.9, 487.9. ¹H NMR (400 MHz, DMSO-d₆): δ8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75(t, J=5.9 Hz, 1H), 4.36 (t, J=5.3 Hz, 2H), 3.58 (dd, J=11.2, 5.6 Hz,2H), 3.18 (s, 3H).

Step C:3-(4-(2-azidoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

To a solution of2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate (2.13 kg, 4.6 mol, containing also some of thecorresponding bromo-compound) in dimethylformamide (4 L) stirring in a22 L flask was added sodium azide (380 g, 5.84 mol). The reaction washeated at 50° C. for 6 h, poured into ice/water (8 L), and extractedwith 1:1 ethyl acetate:heptane (20 L). The organic layer was washed withwater (5 L) and brine (5 L), and the solvents removed in vacuo to affordthe desired product (1464 g, 77%) as a tan solid. LCMS forC₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z=433.0, 435.0. ¹H NMR (DMSO-d₆, 400 MHz): δ8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75(t, J=5.7 Hz, 1H), 3.54 (t, J=5.3 Hz, 2H), 3.45 (dd, J=11.1, 5.2 Hz,2H).

Step D:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

Step D, Part 1: tert-Butyl2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylcarbamate

Sodium iodide (1080 g, 7.2 mol) was added to3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for30 min during which time a mild exotherm was observed.Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution inmethanol (1 L) dropwise at a rate so that the temperature did not exceed35° C., and the reaction was allowed to stir for 3.5 h at ambienttemperature. The reaction was neutralized with 33 wt % solution ofsodium thiosulfate pentahydrate in water (˜1.5 L), diluted with water (4L), and the pH adjusted to 9 carefully with solid potassium carbonate(250 g—added in small portions: watch foaming). Di-tert-butyldicarbonate (318 g, 1.45 mol) was added and the reaction was allowed tostir at room temperature. Additional potassium carbonate (200 g) wasadded in 50 g portions over 4 h to ensure that the pH was still at orabove 9. After stirring at room temperature overnight, the solid wasfiltered, triturated with water (2 L), and then MTBE (1.5 L). A total of11 runs were performed (5.5 kg, 13.38 mol). The combined solids weretriturated with 1:1 THF:dichloromethane (24 L, 4 runs in a 20 L rotaryevaporator flask, 50° C., 1 h), filtered, and washed withdichloromethane (3 L each run) to afford an off-white solid. The crudematerial was dissolved at 55° C. tetrahydrofuran (5 mL/g), treated withdecolorizing carbon (2 wt %) and silica gel (2 wt %), and filtered hotthrough celite to afford the product as an off-white solid (5122 g). Thecombined MTBE, THF, and dichloromethane filtrates were concentrated invacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethylacetate gradient, 30 L) to afford more product (262 g). The combinedsolids of tert-butyl2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylcarbamatewere dried to a constant weight in a convection oven (5385 g, 83%).

Step D, Part 2:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride Method A:

In a 22 L flask was charged hydrogen chloride (4 N solution in1,4-dioxane, 4 L, 16 mol).tert-Butyl[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate(2315 g, 4.77 mol) was added as a solid in portions over 10 min. Theslurry was stirred at room temperature and gradually became a thickpaste that could not be stirred. After sitting overnight at roomtemperature, the paste was slurried in ethyl acetate (10 L), filtered,re-slurried in ethyl acetate (5 L), filtered, and dried to a constantweight to afford the desired product as a white solid (combined withother runs, 5 kg starting material charged, 4113 g, 95%). LCMS forC₁₂H₁₁BrFN₆O₃ (M+H)⁺: m/z=384.9, 386.9. ¹H NMR (400 MHz, DMSO-d₆): δ8.12 (m, 4H), 7.76 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.78 (t, J=6.1 Hz,1H), 3.51 (dd, J=11.8, 6.1 Hz, 2H), 3.02 (m, 2H).

Method B:

tert-Butyl[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate(5000 g) was added to a mixture of isopropanol (20 L) and 4 N HCl in1,4-dioxane (10 L) at room temperature. The batch was heated to 40-45°C. and held for 1 h. Ethyl acetate was added to the batch at 40-45° C.and held for 2.5 h. Upon reaction completion, as indicated by HPLC,heptane (10 L) was added to the batch. The batch was cooled to 25° C.The product was isolated by filtration and the wet cake was washed withethyl acetate (3×5.0 L). The product was dried in a vacuum, oven at 20°C. to give 4344 g (93.4% yield) of the title compound. LC-MS, ¹H and ¹³CNMR, and HPLC data of this lot were identical to those of the productprepared by Method A.

Step E:tert-Butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate[Aldrich, product #142662] (149 mL, 1.72 mol) and dichloromethane (1.5L) and cooled using an ice bath to 2° C. tert-Butanol (162 mL, 1.73 mol)in dichloromethane (200 mL) was added dropwise at a rate so that thetemperature did not exceed 10° C. The resulting solution was stirred atroom temperature for 30-60 min to providetert-butyl[chlorosulfonyl]carbamate.

A 22 L flask was charged with3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After coolingto −15° C. with an ice/salt bath, the solution oftert-butyl[chlorosulfonyl]carbamate (prepared as above) was added at arate so that the temperature did not exceed −10° C. (addition time 7min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) wasadded at a rate so that the temperature did not exceed −5° C. (additiontime 10 min). The cold bath was removed, the reaction was allowed towarm to 10° C., split into two portions, and neutralized with 10% concHCl (4.5 L each portion). Each portion was transferred to a 50 Lseparatory funnel and diluted with ethyl acetate to completely dissolvethe white solid (˜25 L). The layers were separated, and the organiclayer was washed with water (5 L), brine (5 L), and the solvents removedin vacuo to afford an off-white solid. The solid was triturated withMTBE (2×1.5 L) and dried to a constant weight to afford a white solid. Atotal of 4113 g starting material was processed in this manner (5409 g,98%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.90 (s, 1H), 8.08 (dd, J=6.2, 2.5Hz, 1H), 7.72 (m, 1H), 7.59 (t, J=8.6 Hz, 1H), 6.58 (t, J=5.7 Hz, 1H),3.38 (dd, J=12.7, 6.2 Hz, 2H), 3.10 (dd, J=12.1, 5.9 Hz, 2H), 1.41 (s,9H).

Step F:N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

Method A: Using Trifluoroacetic Acid

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) wasaddedtert-butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate(1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixturewas stirred at room temperature for 1.5 h, the solvents removed invacuo, and chased with dichloromethane (2 L). The resulting solid wastreated a second time with fresh 98:2 trifluoroacetic acid:water (8.9L), heated for 1 h at 40-50° C., the solvents removed in vacuo, andchased with dichloromethane (3×2 L). The resulting white solid was driedin a vacuum drying oven at 50° C. overnight. A total of 5409 g wasprocessed in this manner (4990 g, quant. yield). LCMS for C₁₂H₁₂BrFN₇O₅S(M+H)⁺: m/z=463.9, 465.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2,2.5 Hz, 1H), 7.72 (m, 1H), 7.59 (t, J=8.7 Hz, 1H), 6.67 (t, J=5.9 Hz,1H), 6.52 (t, J=6.0 Hz, 1H), 3.38 (dd, J=12.7, 6.3 Hz, 2H), 3.11 (dd,J=12.3, 6.3 Hz).

Method B: Using Hydrochloric Acid

To solution oftert-butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate(4500 g) in isopropanol (9 L) was added 4 N HCl in dioxane (8.0 L). Thereaction mixture was heated to 40-45° C. and was held at thistemperature for about 5 h. Upon completion of reaction (as indicated byHPLC analysis), heptane (72 L) was added to the reaction mixture. Theresultant mixture was heated to 68° C. and held at this temperature for1 h. The batch was allowed to cool to about 23° C. The solid product wascollected by filtration. The wet cake was washed with a mixture ofheptane (16 L) and isopropanol (1.2 L) and dried under suction on afilter funnel. The crude product was dissolved in ethyl acetate (10.8 L)at about 43° C. Heptane (32.4 L) was added to the ethyl acetate solutionover 15 min. The batch was heated to 70° C. and held at this temperaturefor 1 h. The batch was cooled to 21° C. and solid product was collectedby filtration. The wet cake was washed with heptane (14.4 L) and driedunder suction on the filter funnel. Yield of product was 3034 g. LC-MS,¹H and ¹³C NMR, and HPLC data of this lot were identical to those of theproduct prepared by Method A.

Step G:(Z)-4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamideMethod A:

To a crude mixture ofN-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(2.4 mol) containing residual amounts of trifluoroacetic acid stirringin a 22 L flask was added THF (5 L). The resulting solution was cooledto 0° C. using an ice bath and 2 N NaOH (4 L) was added at a rate sothat the temperature did not exceed 10° C. After stirring at ambienttemperature for 3 h (LCMS indicated no starting material remained), thepH was adjusted to 3-4 with concentrated HCl (˜500 mL). The THF wasremoved in vacuo, and the resulting mixture was extracted with ethylacetate (15 L). The organic layer was washed with water (5 L), brine (5L), and the solvents removed in vacuo to afford a solid. The solid wastriturated with MTBE (2×2 L), combined with three other reactions of thesame size, and dried overnight in a convection oven to afford a whitesolid (3535 g). The solid was recrystallized (3×22 L flasks, 2:1deionized ultra-filtered water:ethanol, 14.1 L each flask) and dried ina 50° C. convection oven to a constant weight to furnish the titlecompound as an off-white solid (3290 g, 78%). LCMS for C₁₁H₁₄BrFN₇O₄S(M+H)⁺: m/z=437.9, 439.9. ¹H NMR (400 MHz, DMSO-d₆): δ 11.51 (s, 1H),8.90 (s, 1H), 7.17 (t, J=8.8 Hz, 1H), 7.11 (dd, J=6.1, 2.7 Hz, 1H), 6.76(m, 1H), 6.71 (t, J=6.0 Hz, 1H), 6.59 (s, 2H), 6.23 (t, J=6.1 Hz, 1H),3.35 (dd, J=10.9, 7.0 Hz, 2H), 3.10 (dd, J=12.1, 6.2 Hz, 2H). X-raycrystallographic analysis determined that the title compound adopts aZ-configuration (Z-isomer) with respect to the carbon-nitrogen doublebond (C═N) of oxime functionality.

Method B:

N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(1500 g) was added to THF (6.0 L) and the batch was cooled to 2° C.Trifluoroacetic acid (0.006 L) was added to the batch at 2° C. followedby addition of aqueous sodium hydroxide solution (384 g of solid NaOH in4.8 L of water) at 0-2° C. The batch was warmed up to about 16° C. andheld for 5 h. Upon completion of reaction, as indicated by HPLC,concentrated hydrochloric acid (0.7 L) was added to adjust the pH of thebatch to 3-4. About 4 L of solvent was removed from the batch bydistillation under reduced pressure. The batch was added to ethylacetate (18.0 L) and the biphasic mixture was stirred for 15 min. Theorganic layer was washed with water (6.0 L) and brine (6.0 L)sequentially. The organic solution was dried over anhydrous magnesiumsulfate. Magnesium sulfate was filtered and the filtrate was evaporatedto dryness under reduced pressure. To the resultant solid, MTBE (3.0 L)was added and the slurry was stirred for 15 min. The solid product wasisolated by filtration. The filter cake was washed with MTBE (1.2 L) andheptane (1.2 L) sequentially. The solid was dried on the filter funnelunder suction to give 1416 g (87.9%) of the product. The product (2440g, obtained in two batches) was further purified by re-slurrying in MTBE(9.6 L) at 17° C. for 2 h. The batch was cooled to 6° C. for 30 min. Thesolid product was collected by filtration and the wet cake was washedwith MTBE (3.6 L) and heptane (1.2 L) sequentially. The product wasdried in a vaccum oven at 20° C. to give 1962 g of the title compound in81.7% yield. LC-MS, ¹H and ¹³C NMR, and HPLC data of this lot wereidentical to those of the product prepared by Method A.

Example 24 Compound Data

Select physical and biological activity data for the compounds ofExample 1-19 are summarized in Table 2 below. 1050 data are from theassay provided in Example A.

TABLE 2

Ex. IDO MS No. R₁ R₂ R₃ n IC₅₀ (nM) [M + H] 1 NH₂ Br F 1 <200 437.9,439.9 2 Me Br F 1 <200 437.0, 439.0 3 NH₂ Br F 2 <100 451.8, 453.9 4 MeBr F 2 <100 451.0, 453.0 5 NH₂ Cl F 1 <200 394.0 6 Me Cl F 1 <200 393.07 NH₂ Cl F 2 <200 408.1 8 Me Cl F 2 <200 407.1 9 NH₂ CF₃ F 1 <100 428.010 Me CF₃ F 1 <100 427.0 11 NH₂ CF₃ F 2 <100 442.0 12 Me CF₃ F 2 <100441.1 13 NH₂ CF₃ H 1 <500 410.0 14 Me CF₃ H 1 <200 409.1 15 NH₂ CF₃ H 2<200 424.0 16 Me CF₃ H 2 <200 423.1 17 Me CH₃ F 1 <500 373.1 18 NH₂ CN F1 <750 385.0 19 Me CN F 1 <500 406.0* *[M + Na]

Example 25 Compound Data

IDO IC₅₀ data (see Example A) for the compounds of Examples 20 and 21 isprovided below in Table 3.

TABLE 3 Ex. No. IDO IC₅₀ (nM) 20 <500 21 <750

Example 26 NMR Data

¹H NMR data (Varian Inova 500 spectrometer, a Mercury 400 spectrometer,or a Varian (or Mercury) 300 spectrometer) for the compounds of Examples1-21 is provided below in Table 4.

TABLE 4 Ex. No. Solvent MHz ¹H NMR Spectra 1 DMSO-d₆ 400 δ 11.5 (s, 1H), 8.89 (s, 1 H), 7.17 (dd, J = 8.8, 8.6 Hz, 1 H), 7.09 (dd, J = 6.1,2.7 Hz, 1 H), 6.76-6.72 (m, 1 H), 6.56 (dd, J = 6.1, 6.1 Hz, 1 H), 6.51(s, 2 H), 6.17 (dd, J = 5.9, 5.9 Hz, 1 H), 3.27- 3.21 (m, 2 H),2.94-2.88 (m, 2 H), 1.78-1.71 (m, 2 H) 2 DMSO-d₆ 400 δ 11.49 (s, 1H),8.90 (s, 1H), 7.17 (m, 2H), 7.09 (dd, J = 6.3, 2.5 Hz, 1H), 6.26 (t, J =6.1 Hz, 1H), 3.33 (m, 2H), 3.13 (q, J = 6.0 Hz, 2H), 2.89 (s, 3H) 3DMSO-d₆ 400 δ 11.5 (s, 1 H), 8.89 (s, 1 H), 7.17 (dd, J = 8.8, 8.6 Hz, 1H), 7.09 (dd, J = 6.1, 2.7 Hz, 1 H), 6.76-6.72 (m, 1 H), 6.56 (dd, J =6.1, 6.1 Hz, 1 H), 6.51 (s, 2 H), 6.17 (dd, J = 5.9, 5.9 Hz, 1 H), 3.27-3.21 (m, 2 H), 2.94-2.88 (m, 2 H), 1.78-1.71 (m, 2 H) 4 CD₃OD 400 δ 7.12(dd, J = 5.9, 2.4 Hz, 1H), 7.05 (t, J = 8.7 Hz, 1H), 6.83 (m, 1H), 3.39(t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.6 Hz, 2H), 2.94 (s, 3H), 1.87 (m,2H) 5 DMSO-d₆ 400 δ 7.96 (dd, J = 6.8, 2.1 Hz, 0.05 H), 7.32-7.29 (m,0.1 H), 7.18 (dd, J = 9.1, 9.1 Hz, 0.95 H), 6.93 (dd, J = 6.4, 2.7 Hz,0.95 H), 6.71-6.66 (m, 0.95 H), 6.33 (br s, 1 H), 3.35-3.27 (m, 2 H),3.10- 3.06 (m, 2 H) 6 DMSO-d₆ 400 δ 11.50 (s, 1H), 8.91 (s, 1H), 7.19(m, 2H), 6.96 (dd, J = 6.7, 2.5 Hz, 1H), 6.71 (m, 1H), 6.26 (t, J = 6.4Hz, 1H), 3.32 (m, 2H), 3.13 (q, J = 5.8 Hz, 2H), 2.89 (s, 3H) 7 DMSO-d₆400 δ 8.90 (s, 1 H), 7.20 (dd, J = 9.2, 9.0 Hz, 1 H), 6.96 (dd, J = 6.4,2.7 Hz, 1 H), 6.72-6.69 (m, 1 H), 6.55 (t, J = 6.0 Hz, 1 H), 6.51 (s, 2H), 6.16 (t, J = 5.9 Hz, 1 H), 3.28-3.21 (m, 2 H), 2.93-2.87 (m, 2H),1.76-1.72 (m, 2 H) 8 CD₃OD 300 δ 7.06 (t, J = 8.9 Hz, 1H), 6.98 (m, 1H),6.80 (m, 1H), 3.73 (m, 2H), 3.28 (m, 2H), 2.94 (s, 3H), 1.28 (m, 2H) 9DMSO-d₆ 400 δ 11.60 (s, 1H), 9.06 (s, 1H), 7.30 (t, J = 10.1 Hz, 1H),7.14 (dd, J = 6.1, 2.7 Hz, 1H), 7.03 (m, 1H), 6.71 (t, J = 5.3 Hz, 1H),6.58 (s, 2H), 6.23 (t, J = 6.2 Hz, 1H), 3.36 (q, J = 6.5 Hz, 2H), 3.08(m, 2H) 10 DMSO-d₆ 400 δ 11.60 (s, 1H), 9.07 (s, 1H), 7.30 (t, J = 10.1Hz, 1H), 7.18 (t, J = 6.0 Hz, 1H), 7.13 (dd, J = 6.0, 2.7 Hz, 1H), 7.03(m, 1H), 6.27 (t, J = 6.3 Hz, 1H), 3.32 (m, 2H), 3.13 (q, J = 6.0 Hz,2H), 2.89 (s, 3H) 11 DMSO-d₆ 300 δ 11.6 (s, 1 H), 9.08 (s, 1 H), 7.31(dd, J = 10.0, 9.4 Hz, 1 H), 7.13 (dd, J = 6.4, 2.9 Hz, 1 H), 7.05-6.99(m, 1 H), 6.58 (t, J = 6.0 Hz, 1 H), 6.52 (s, 2 H), 6.17 (t, J = 5.9 Hz,1 H), 3.28-3.21 (m, 2 H), 2.94-2.87 (m, 2 H), 1.79-1.72 (m, 2 H) 12DMSO-d₆ 400 δ 11.6 (s, 1 H), 9.07 (s, 1 H), 7.30 (dd, J = 10.0, 9.6 Hz,1 H), 7.13 (dd, J = 6.2, 2.5 Hz, 1 H), 7.05-7.02 (m, 2 H), 6.19 (t, J =5.8 Hz, 1 H), 3.27-3.21 (m, 2 H), 2.99-2.94 (m, 2 H), 2.87 (s, 3 H),1.76- 1.72 (m, 2 H) 13 CD₃OD 400 δ 7.36 (t, J = 7.8 Hz, 1H), 7.23 (d, J= 7.8 Hz, 1H), 7.10 (s, 1H), 7.03 (d, J = 7.8 Hz, 1H), 3.48 (m, 2H),3.29 (m, 2H) 14 DMSO-d₆ 500 δ 11.63 (s, 1H), 9.08 (s, 1H), 7.39 (t, J =7.6 Hz, 1H), 7.21 (m, 2H), 7.10 (s, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.28(t, J = 5.4 Hz, 1H), 3.36 (q, J = 5.8 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H),2.91 (s, 3H) 15 DMSO-d₆ 400 δ 11.6 (s, 1 H), 9.12 (s, 1 H), 7.37 (dd, J= 8.0, 8.0 Hz, 1 H), 7.21- 7.18 (m, 1 H), 7.07 (s, 1 H), 6.95 (d, J =10.0 Hz, 1 H), 6.52 (br s, 3 H), 6.17 (t, J = 6.0 Hz, 1 H), 3.28-3.22(m, 2 H), 2.93-2.89 (m, 2 H), 1.77-1.73 (m, 2 H) 16 DMSO-d₆ 400 δ 11.6(s, 1 H), 9.11 (s, 1 H), 7.37 (dd, J = 8.0, 8.0 Hz, 1 H), 7.20 (d, J =7.8 Hz, 1 H), 7.07-7.01 (m, 2 H), 6.96 (d, J = 8.0 Hz, 1 H), 6.20 (t, J= 5.9 Hz, 1 H), 3.27-3.22 (m, 2 H), 2.99-2.94 (m, 2 H), 2.87 (s, 3 H),1.78-1.71 (m, 2 H) 17 DMSO-d₆ 400 δ 11.25 (s, 1H), 8.61 (s, 1H), 7.18(m, 1H), 6.91 (m, 1H), 6.72 (m, 1H), 6.58 (m, 1H), 6.24 (s, 2H), 3.32(m, 2H), 3.11 (m, 2H), 2.89 (s, 3H), 2.05 (s, 3H). 18 DMSO-d₆ 400 δ11.65 (s, 1H), 9.08 (s, 1H), 7.34 (t, J = 9.1 Hz, 1H), 7.22 (dd, J =5.4, 2.8 Hz, 1H), 7.13 (m, 1H), 6.70 (t, J = 5.9 Hz, 1H), 6.59 (s, 2H),6.20 (t, J = 6.1 Hz, 1H), 3.34 (m, 2H), 3.09 (m, 2H) 19 DMSO-d₆ 400 δ11.65 (s, 1H), 9.08 (s, 1H), 7.35 (m, 1H), 7.18 (m, 3H), 6.56 (m, 1H),6.23 (m, 1H), 6.24 (s, 2H), 3.32 (m, 2H), 3.14 (m, 2H), 2.89 (s, 3H). 20DMSO-d₆ 400 δ 10.87 (s, 1 H), 7.75 (s, 1 H), 6.83 (t, J = 7.3 Hz, 1 H),6.68 (t, J = 6.0 Hz, 1 H), 6.56 (s, 2 H), 6.30 (t, J = 6.0 Hz, 1 H),6.23 (s, 1 H), 4.56 (d, J = 7.0 Hz, 2 H), 3.32 (q, J = 6.3 Hz, 2 H),3.07 (q, J = 6.3 Hz, 2 H) 21 DMSO-d₆ 400 δ 10.88 (s, 1 H), 7.77 (s, 1H), 6.83 (t, J = 6.8 Hz, 1 H), 6.68 (t, J = 5.9 Hz, 1 H), 6.56 (s, 2 H),6.30 (t, J = 5.9 Hz, 1 H), 6.22 (s, 1 H), 4.55 (d, 2 H), 3.32 (q, J =6.3 Hz, 2 H), 3.06 (q, J = 6.3 Hz, 2 H)

Example A Human Indoleamine 2,3-Dioxygenasae (IDO) Enzyme Assay

Human indoleamine 2,3-dioxygenasae (IDO) with an N-terminal His tag wasexpressed in E. coli and purified to homogeneity. IDO catalyzes theoxidative cleavage of the pyrrole ring of the indole nucleus oftryptophan to yield N′-formylkynurenine. The assays were performed atroom temperature as described in the literature using 95 nM IDO and 2 mMD-Trp in the presence of 20 mM ascorbate, 5 μM methylene blue and 0.2mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initialreaction rates were recorded by continuously following the absorbanceincrease at 321 nm due to the formation of N′-formlylkynurenine (See:Sono, M., et al., 1980, J. Biol. Chem. 255, 1339-1345).

Example B Determination of Inhibitor Activity in HeLa Cell-BasedIndoleamine 2,3-Dioxygenase (IDO)/Kynurenine Assay

HeLa cells (#CCL-2) were obtained from the American Type Tissue CultureCollection (ATCC, Manassas, Va.) and routinely maintained in minimumessential medium (eagle) with 2 mM L-glutamine and Earle's BSS adjustedto contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids,1 mM sodium pyruvate and 10% fetal bovine serum (all from Invitrogen).Cells were kept at 37° C. in a humidified incubator supplied with 5%CO₂. The assay was performed as follows: HeLa cells were seeded in a 96well culture plate at a density of 5×10³ per well and grown overnight.On the next day, IFN-γ (50 ng/mL final concentration) and serialdilutions of compounds (in total volume of 200 μL culture medium) wereadded into cells. After 48 hours of incubation, 140 μL of thesupernatant per well was transferred to a new 96 well plate. 10 μL of6.1 N trichloroacetic acid (#T0699, Sigma) was mixed into each well andincubated at 50° C. for 30 min to hydrolyze N-formylkynurenine producedby indoleamine 2,3-dioxygenase to kynurenine. The reaction mixture wasthen centrifuged for 10 min at 2500 rpm to remove sediments. 100 μL ofthe supernatant per well was transferred to another 96 well plate andmixed with 100 μL of 2% (w/v)p-dimethylaminobenzaldehyde (#15647-7,Sigma-Aldrich) in acetic acid. The yellow color derived from Kynureninewas measured at 480 nm using a SPECTRAmax 250 microplate reader(Molecular Devices). L-kynurenine (#K8625, Sigma) was used as standard.The standards (240, 120, 60, 30, 15, 7.5, 3.75, 1.87 μM) were preparedin 100 μL culture media and mixed with equal volume of 2% (w/v)p-dimethylaminobenzaldehyde. The percent inhibition at individualconcentrations was determined and the average values of duplicates wereobtained. The data was analyzed by using nonlinear regression togenerate IC₅₀ values (Prism Graphpad). See: Takikawa 0, et al., 1988, JBiol. Chem., 263(4): 2041-8.

Example C Determination of Effect of IDO Inhibitors on T CellProliferation that is Suppressed by IDO-Expressing Dendritic Cells

Monocytes were collected from human peripheral mononuclear cells byleukophoresis. Monocytes were then seeded at a density of 1×10⁶cells/well in a 96 well plate, using RPMI 1640 medium supplemented with10% fetal bovine serum and 2 mM L-glutamine (all from Invitrogen).Adherent cells were retained on the plate after overnight culture at 37°C. Adherent monocytes were then stimulated for 5-7 days with 100 ng/mlGM-CSF (#300-03, PeproTech) and 250 ng/ml IL-4 (#200-04, PeproTech),followed by activation with 5 μg/mL LPS from Salmonella typhimurium(#437650, Sigma) and 50 ng/mL IFN-γ (#285-IF, R&D Systems) foradditional 2 days to induce dendritic cell maturation.

After dendritic cell activation, the medium was replaced with completedRPMI 1640 supplemented with 100-200 U/mL IL-2 (#CYT-209, ProSpec-TanyTechnoGene) and 100 ng/mL anti-CD3 antibody (#555336, PharMingen), Tcells (2-3×10⁵ cells/well), and serial dilutions of IDO compounds. Afterincubation for 2 more days, T cell proliferation was measured by BrdUincorporation assay, using a colorimetric Cell Proliferation ELISA kitper manufacturer's instruction (#1647229, Roche Molecular Biochemicals).Cells were continuously cultured for 16-18 hrs in presence of 10 μM BrdUlabeling solution. Then, the labeling medium was removed, and 200 μLFixDenat per well was added to the cells and incubated for 30 minutes atroom temperature. The FixDenat solution was removed and 100 μL/wellanti-BrdU-POD antibody conjugate working solution was added. Thereaction was carried out for 90 minutes at room temperature. Theantibody conjugate was then removed, and cells were rinsed three timeswith 200 μL/well washing solution. Finally, 100 μL/well of substratesolution was added and the results were obtained using a microplatereader (Spectra Max PLUS, Molecular Devices) during color development.Multiple readings at various time points were obtained to ensure thedata was within the linear range. The data was routinely obtained fromreplicated experiments, and appropriate controls were included. See:Terness P, et al. 2002, J. Exp. Med., 196(4): 447-57; and Hwu, P, et al.2000, J. Immunol., 164(7): 3596-9.

Example D In Vivo Testing of IDO Inhibitors for Antitumor Activity

In vivo anti-tumor efficacy can be tested using modified tumorallograft/xenograft protocols. For instance, it has been described inthe literature that IDO inhibition can syngerize with cytotoxicchemotherapy in immune-competent mice (Muller, A. J., et al. 2005, Nat.Med. 11:312-319). This synergy was shown to be dependent on T-cells bycomparison of the synergistic effects of an investigational IDOinhibitor in murine tumor xenograft models (e.g. B16 and relatedvariants, CT-26, LLC) grown in immune competent syngenic mice to thatobserved in syngenic mice treated with neutralizing anti-CD4 antibodies,or the same tumors grown in immune-compromised mice (e.g. nu/nu).

The concept of differential anti-tumor effects in immune-competentversus immune-compromised mice may also permit testing ofinvestigational IDO inhibitors as single agents. For instance, LLCtumors grow well in their syngenic host strain, C57Bl/6. However, ifthese mice are treated with the IDO inhibitor 1-MT (versus placebo) theformation of tumors is markedly delayed, implying that IDO inhibitionwas growth inhibitory (Friberg, M., et al. 2002, Int. J. Cancer101:151-155). Following this logic, one can examine the efficacy of IDOinhibition in the LLC xenograft tumor model grown in C57Bl/6 immunecompetent mice and compare that to the effects of IDO inhibitors on LLCtumor growth in nude or SCID mice (or C57Bl/6 mice treated withantibodies that neutralize T-cell activity). As the effects of relievingthe tumor-mediated immune suppressive activity of IDO will likely differdepending on the immunogenic potential of different tumor models,genetic modifications can be made to the tumor cells to increase theirimmunogenic potential. For instance, expression of GM-CSF in B16.F10cells increases their immunogenic potential (Dranoff, G., et al. 1993,Proc. Natl. Acad. Sci., USA, 90:3539-3543). As such, in some tumormodels (e.g. B16.F10) one can generate [poly]clones that express immunestimulatory proteins such as GM-CSF and test the growth inhibitoryeffects of IDO inhibitors against tumors established from these tumorcells in both immune-competent and -compromised mice.

A third avenue for assessing the efficacy of IDO inhibitors in vivoemploys ‘pre-immunization’ murine tumor allograft/xenograft models. Inthese models, immune-competent mice are sensitized to a specific tumorantigen or antigens to mimic a therapeutic anti-tumor vaccination. Thisprimes the mice for an anti-tumor response mediated by the immune systemwhen mice are subsequently challenged with murine tumor cell lines(possessing similar tumor antigens to those used for immunization) inxenograft experiments. Expression of IDO has been shown to blunt theanti-tumor response and allow xenografts to grow more rapidly.Importantly, the growth of tumors in this model is inhibited by the IDOinhibitor 1-MT (Uyttenhove, C., et al. 2003, Nat. Med. 9:1269-1274).This model is particularly attractive as IDO activity is permissive forP815 tumor growth and specific inhibition of IDO should therefore growthinhibitory.

Lastly, therapeutic immunization may be used to evaluate the impact ofIDO inhibitors in vivo. For example, it has been demonstrated usingB16-BL6 cells that one can challenge Blk/6 mice with an intravenousinjection of tumor cells followed by treatment with a well characterizedimmunogenic peptide (e.g. TRP-2) expressed by the tumor cells (Ji, etal., 2005, J. Immunol, 175: 1456-63). Importantly, immune systemmodifiers, such as anti-CTL-4 antibody, can improve responses to suchtherapeutic immunizations. The impact of IDO inhibitors may be evaluatedin a similar manner—tumor peptide immunization with or without IDOinhibitor. Efficacy is assess by animal survival (time to morbidity) orby the measurement of tumor metastases to the lungs and/or other organsat defined timepoints.

In any/all of the above mentioned models, it may also be possible todirectly and/or indirectly measure the number and/or activity of tumorreactive immune cells. Methods for measuring the number and/or activityof tumor reactive immune cells are well established and can be performedusing techniques familiar to those schooled in the art (CurrentProtocols in Immunology, Vol. 4, Coligan, J. E., et al.; Immunotherapyof Cancer, Human Press, 2006, Disis, M. L. and references therein).Conceptually, a reduction in the immune suppressive effects of IDO mayresult in increased numbers or reactivity of tumor specific immunecells. Further, IDO inhibition may further increase the number orreactivity of tumor reactive immune cells when combined with othertherapeutics, for example chemotherapeutics and/or immune modulators(e.g. anti-CTLA4 antibody).

All allograft/xenograft experiments can be performed using standardtumor techniques (reviewed by Corbett, et al., In Cancer Drug Discoveryand Development: Anticancer Drug Development Guide: PreclinicalScreening, Clinical Trials, and Approval, 2^(nd) Ed. Teicher, B. A. andAndrews, P. A., Gumana Press Inc.: Totowa, N.J., 2004). The cloning andintroduction of genes (e.g. IDO, GM-CSF) into tumor cell lines, can beperformed using techniques familiar to those schooled in the art(reviewed in Sambrook, J. and Russel, D., Molecular Cloning: Alaboratory Manual (3^(rd) edition), Cold Spring Harbor Laboratory Press:Cold Spring Harbor, N.Y., 2001).

Example E In Vivo Testing of IDO Inhibitors in Human ImmunodeficiencyVirus-1 (HIV-1) Encephalitis Model 1. Cell Isolation and Viral Infection

Monocytes and PBL can be obtained by countercurrent centrifugalelutriation of leukopheresis packs from HIV-1, 2 and hepatitis Bseronegative donors. Monocytes are cultivated in suspension cultureusing Teflon flasks in Dulbecco's Modified Eagle's Medium (DMEM,Sigma-Aldrich) supplemented with 10% heat-inactivated pooled humanserum, 1% glutamine, 50 μg/mL gentamicin, 10 μg/mL ciprofloxacin(Sigma), and 1000 U/mL highly purified recombinant human macrophagecolony stimulating factor. After seven days in culture, MDM are infectedwith HIV-1ADA at multiplicity of infection of 0.01.

2. Hu-PBL-NOD/SCID HIVE Mice

Four-wk old male NOD/C.B-17 SCID mice can be purchased (JacksonLaboratory). Animals are maintained in sterile microisolator cages underpathogen-free conditions. All animals are injected intraperitoneallywith rat anti-CD122 (0.25 mg/mouse) three days before PBLtransplantation and twice with rabbit asialo-GM1 antibodies (0.2mg/mouse) (Wako) one day before and three days after PBL injection(20×10⁶ cells/mouse). HIV-1_(ADA)-infected MDM (3×10⁵ cells in 10 μL)are injected intracranially (i.c.) eight days following PBLreconstitution generating hu-PBL-NOD/SCID HIVE mice. Immediatelyfollowing i.c. injection of HIV-1 infected MDM the hu-PBL-NOD/SCID HIVEmice are subcutaneously (s.c) implanted with control (vehicle) orcompound pellets (14 or 28 day slow release, Innovative Research).Initial experiments are designed to confirm the induction ofvirus-specific CTL in the hu PBL-NOD/SCID HIVE animals treated with IDOcompounds. This is confirmed by tetramer staining and neuropathologicanalyses of MDM elimination from the brain tissue. Then, the experimentis designed to analyze human lymphocyte reconstitution, humoral immuneresponses, and neuropathological alterations. In these experiments,animals are bled on day 7 and sacrificed at 14 and 21 days after i.c.injection of human MDM. Blood collected in EDTA-containing tubes is usedfor flow cytometry and plasma is used for detection of HIV-1 p24 usingELISA (Beckman Coulter™). HIV-1-specific antibodies are detected byWestern blot tests according to the manufacturer instructions (CambridgeBiotech HIV-1 Western blot kit, Calypte Biomedical). Similar amount ofvirus-specific antibodies are detected in control and compound-treatedanimals. A total of three independent experiments can be performed usingthree different human leukocyte donors.

3. FACScan of Peripheral Blood and Spleen in Hu PBL-NOD/SCID HIVE Mice

Two-color FACS analysis can be performed on peripheral blood at wk 1-3and splenocytes at wk 2 and 3 after i.c. injection of human MDM. Cellsare incubated with fluorochrome-conjugated monoclonal Abs (mAbs) tohuman CD4, CD8, CD56, CD3, IFN-γ (eBioscience) for 30 min at 4° C. Toevaluate the cellular immune response, IFN-γ intracellular staining isperformed in combination with anti-human CD8 and FITC-conjugatedanti-mouse CD45 to exclude murine cells. To determine the Ag-specificCTL, allophycocyanin-conjugated tetramer staining for HIV-1^(gag) (p17(aa77-85) SLYNTVATL, SL-9) and HIV-1^(pol) [(aa476-485) ILKEPVHGV, IL-9]is performed on phytohemaglutinin/interleukin-2 (PHA/IL-2)-stimulatedsplenocytes. Cells are stained following the recommendation of theNIH/National Institute of Allergy and Infections Disease, NationalTetramer Core Facilities. Data were analyzed with a FACS Calibur™ usingCellQuest software (Becton Dickinson Immunocytometry System).

4. Histopathology and Image Analyses

Brain tissue is collected at days 14 and 21 after i.c. injection of MDM,fixed in 4% phosphate-buffered paraformaldehyde and embedded in paraffinor frozen at −80° C. for later use. Coronal sections from the embeddedblocks are cut in order to identify the injection site. For each mouse,30-100 (5-μm-thick) serial sections are cut from the human MDM injectionsite and 3-7 slides (10 sections apart) are analyzed. Brain sections aredeparaffinized with xylene and hydrated in gradient alcoholsImmunohistochemical staining follows a basic indirect protocol, usingantigen retrieval by heating to 95° C. in 0.01 mol/L citrate buffer for30 min for antigen retrieval. To identify human cells in mouse brains,mAb to vimentin (1:50, clone 3B4, Dako Corporation), which identifiesall human leukocytes is used. Human MDM and CD8⁺ lymphocytes aredetected with CD68 (1:50 dilution, clone KP 1) and CD8 (1:50 dilution,clone 144B) antibodies, respectively. Virus-infected cells are labeledwith mAb to HIV-1 p24 (1:10, clone Kal-1, all from Dako). Reactivemurine microglial cells are detected with Iba-1 antibody (1:500, Wako).Expression of human IDO (huIDO) is visualized with Abs obtained from theDepartment of Cell Pharmacology, Central Research Institute, GraduateSchool of Medicine, Hokkaido University, Sapporo, Japan. Primaryantibodies are detected with the appropriate biotinylated secondaryantibodies and visualized with avidin-biotin complexes (Vectastain EliteABC kit, Vector Laboratories) and horseradish peroxidase (HRP) coupleddextran polymer (EnVision, Dako Corporation) Immunostained sections arecounterstained with Mayer's hematoxylin. Sections from which primaryantibody is deleted or irrelevant IgG isotype is incorporated served ascontrols. Two independent observers in a blinded fashion count thenumbers of CD8⁺ lymphocytes, CD68⁺ MDM and HIV-1 p24⁺ cells in eachsection from each mouse. Light microscopic examination is performed witha Nikon Eclipse 800 microscope (Nikon Instruments Inc).Semi-quantitative analysis for Ibal (percentage of area occupied byimmunostaining) is carried out by computer-assisted image analysis(Image-Pro®Plus, Media Cybernetics) as previously described.

5. Statistical Analysis

Data can be analyzed using Prism (Graph Pad) with Student t-test forcomparisons and ANOVA. P-values <0.05 were considered significant.

6. Reference

-   Poluektova L Y, Munn D H, Persidsky Y, and Gendelman H E (2002).    Generation of cytotoxic T cells against virus-infected human brain    macrophages in a murine model of HIV-1 encephalitis. J. Immunol.    168(8):3941-9.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including all patent,patent applications, and publications, cited in the present applicationis incorporated herein by reference in its entirety.

1-174. (canceled)
 175. A pharmaceutical composition comprising acompound of the formula:

and at least one pharmaceutically acceptable carrier.
 176. Thepharmaceutical composition of claim 175, wherein the composition is aunit dosage form.
 177. The pharmaceutical composition of claim 176,wherein the unit dosage form is a tablet.
 178. The pharmaceuticalcomposition of claim 176, wherein the unit dosage form is a capsule.179. A pharmaceutically acceptable salt of the compound of formula:


180. A pharmaceutical composition comprising the pharmaceuticallyacceptable salt of claim 179 and at least one pharmaceuticallyacceptable carrier.
 181. The pharmaceutical composition of claim 180,wherein the composition is a unit dosage form.
 182. The pharmaceuticalcomposition of claim 181, wherein the unit dosage form is a tablet. 183.The pharmaceutical composition of claim 181, wherein the unit dosageform is a capsule.